High molecular weight polymer-based prodrugs

ABSTRACT

The present invention is directed compositions of the formula: ##STR1## wherein: D is a residue of a biologically active moiety; 
     X is an electron withdrawing group; 
     Y and Y&#39; are independently O or S; 
     (n) is zero (0) or a positive integer, preferably from 1 to about 12; 
     R 1  and R 2  are independently selected from the group consisting of H, C 1-6  alkyls, aryls, substituted aryls, aralkyls, heteroalkyls, substituted heteroalkyls and substituted C 1-6  alkyls; 
     R 3  is a substantially non-antigenic polymer, C 1-12  straight or branched alkyl or substituted alkyl, C 5-8  cycloalkyl or substituted cycloalkyl, carboxyalkyl, carboalkoxy alkyl, dialkylaminoalkyl, phenylalkyl, phenylaryl or ##STR2## R 4  and R 5  are independently selected from the group consisting of H, C 1-6  alkyls, aryls, substituted aryls, aralkyls, heteroalkyls, substituted heteroalkyls, and substituted C 1-6  alkyls or jointly form a cyclic C 5  -C 7  ring. 
     In preferred embodiments, the prodrugs contain a polyethylene glycol having a molecular weight of at least about 20,000.

This application is a continuation of U.S. patent application Ser. No.08/914,927 now U.S. Pat. No. 5,965,566 filed Aug. 20, 1997 which, inturn, is a continuation-in-part of U.S. patent application Ser. No.08/700,269 filed Aug. 20, 1996, now U.S. Pat. No. 5,840,900 which, inturn, is a continuation-in-part of U.S. patent application Ser. No.08/537,207 filed Sep. 29, 1995, now U.S. Pat. No. 5, 880,131 which, inturn, is a continuation-in-part of U.S. patent application Ser. No.08/380,873 filed Jan. 30, 1995, now U.S. Pat. No. 5,614,549 which, inturn, is a continuation-in-part of U.S. patent application Ser. No.08/140,346 filed Oct. 20, 1993 now abandoned. The contents of each ofthe foregoing applications are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to water soluble prodrugs. In particular,the invention relates to the use of relatively high molecular weightnon-antigenic polymers to prepare prodrugs.

BACKGROUND OF THE INVENTION

Over the years, several methods of administering biologically-effectivematerials to mammals have been proposed. Many medicinal agents areavailable as water-soluble salts and can be included in pharmaceuticalformulations relatively easily. Problems arise when the desiredmedicinal is either insoluble in aqueous fluids or is rapidly degradedin vivo. Alkaloids are often especially difficult to solubilize.

For example, several methods have been suggested to overcome theproblems associated with administering paclitaxel, (also known asTaxol®, Bristol-Myers Squibb Co. NY, N.Y.), which is insoluble in water.Currently, paclitaxel is administrated in physical admixture with anon-aqueous vehicle, cremophor-EL. This formulation, however, hasseveral drawbacks. Hypersensitivity reactions have been associated withthe vehicle and intravenous administration of the agent with thisvehicle is also slow and causes discomfort to the patient.

Several methods have been suggested to enhance the aqueous solubility ofpaclitaxel. See, for example, PCT WO 93/24476, U.S. Pat. No 5,362,831,and Nicolaou, et al. Angew. Chem. Int. Ed. Engl. (1994) 33, No. 15/16,pages 1583-1587. Preparing water-soluble prodrug versions has also beenexplored.

Prodrugs include chemical derivatives of a biologically-active parentcompound which, upon administration, will eventually liberate the activeparent compound in vivo. Use of prodrugs allows the artisan to modifythe onset and/or duration of action in vivo. In addition, the use ofprodrugs can modify the transportation, distribution or solubility of adrug in the body. Furthermore, prodrugs may reduce the toxicity and/orotherwise overcome difficulties encountered when administeringpharmaceutical preparations.

A typical example in the preparation of prodrugs can involve conversionof alcohols or thioalcohols to either organic phosphates or esters.Remington's Pharmaceutical Sciences, 16th Ed., A. Osol, Ed. (1980), thedisclosure of which is incorporated by reference herein.

Prodrugs are often biologically inert or substantially inactive forms ofthe parent or active compound. The rate of release of the active drug isinfluenced by several factors including the rate of hydrolysis of theconverted ester or other functionality.

Recently, polyethylene glycol (PEG) and related polyalkylene oxides(PAO's) have been suggested as possible adjuncts for the preparation ofpaclitaxel prodrugs. See PCT WO 93/24476 supra, for example. PEG hasalso been conjugated to proteins, peptides and enzymes to increaseaqueous solubility and circulating life in vivo as well as reduceantigenicity. See, for example, U.S. Pat. Nos. 5,298,643 and 5,321,095,both to Greenwald, et al. These latter two references disclose, interalia, biologically-active conjugates having substantiallyhydrolysis-resistant bonds (linkages) between a polyalkylene oxide andthe target moiety. Thus, long-lasting conjugates rather than prodrugsper se were prepared. In most situations, the average molecular weightof the polymer included in the conjugate was preferably about 5,000daltons.

PCT WO 93/24476 discloses using an ester linkage to covalently bindpaclitaxel to water-soluble polyethylene glycols and provide a prodrug.Applicants, however, have discovered that the ester linkages describedtherein provide T_(1/2) for hydrolysis of greater than four days inaqueous environments. Thus, most of the conjugate is eliminated prior tohydrolysis being achieved in vivo. It would be preferable to provide anester linkage which allows more rapid hydrolysis of the polymer-druglinkage in vivo so as to generate the parent drug compound more rapidly.

It has also been surprisingly found that when only one or two polymersof less than 10,000 molecular weight are conjugated to alkaloids and/ororganic compounds, the resulting conjugates are rapidly eliminated invivo. In fact, such conjugates are so rapidly cleared from the body thateven if a hydrolysis-prone ester linkage is used, not enough of theparent molecule is regenerated in vivo to make the PAO-drug conjugateworthwhile as a prodrug.

Ohya, et al., J. Bioactive and Compatible Polymers Vol. 10 January,1995, 51-66, disclose doxorubicin-PEG conjugates which are prepared bylinking the two substituents via various linkages including esters. Themolecular weight of the PEG used, however, is only about 5,000 at best.Thus, the true in vivo benefits would not be realized because theconjugates would be substantially excreted prior to sufficienthydrolysis of the linkage to generate the parent molecules.

Yamaoka, et al. J. Pharmaceutical Sciences, Vol. 83, No. 4, April 1994,pages 601-606, disclose that the half-life of unmodified PEG incirculation of mice after IV administration extended from 18 minutes toone day when molecular weight was increased from 6,000 to 190,000.Yamaoka, et al., however, failed to consider the effect of linking thepolymer to a drug would have on the drug. Also, Yamaoka, et al. failedto consider that aqueous solutions of higher molecular weight polymersare quite viscous and difficult to dispense through the narrow-boredevices used to administer pharmaceutical preparations.

U.S. Pat. No. 4,943,579 discloses the use of certain amino acid estersin their salt forms as water soluble prodrugs. The reference does not,however, disclose using the amino acids as part of a linkage which wouldattach relatively high molecular weight polymers in order to formprodrugs. As evidenced by the data provided in Table 2 of the '579patent, hydrolysis is quick. At physiologic pH, the insoluble base israpidly generated after injection, binds to proteins and is quicklyeliminated from the body before therapeutic effect can be achieved.

In summary, previous prodrugs based on conjugates of a parent drugcompound and a water soluble polymer have not been successful forvarious reasons including excessively slow hydrolysis of the polymerfrom the parent drug and excessively rapid clearance of the prodrug fromthe body. In addition, improvements in prodrugs based on simple aminoacid esters have been sought to overcome the rapid regeneration of theparent compound at physiological pH.

The present invention addresses the shortcomings described above.

SUMMARY OF THE INVENTION

In one aspect of the invention, compositions of formula (I) areprovided: ##STR3## wherein: D is a residue of a biologically activemoiety;

X is an electron withdrawing group;

Y and Y' are independently O or S;

(n) is zero (0) or a positive integer, preferably from 1 to about 12;

R₁ and R₂ are independently selected from the group consisting of H C₁₋₆alkyls, aryls, substituted aryls, aralkyls, heteroalkyls, substitutedheteroalkyls and substituted C₁₋₆ alkyls;

R₃ is a substantially non-antigenic polymer, C₁₋₁₂ straight or branchedalkyl or substituted alkyl, C₅₋₈ cycloalkyl or substituted cycloalkyl,carboxyalkyl, carboalkoxy alkyl, dialkylaminoalkyl, phenylalkyl,phenylaryl or ##STR4## R₄ and R₅ are independently selected from thegroup consisting of H, C₁₋₆ alkyls, aryls, substituted aryls, aralkyls,heteroalkyls, substituted heteroalkyls, and substituted C₁₋₆ alkyls orjointly form a cyclic C₅ -C₇ ring.

In those aspects of the invention where R₃ is not a substantiallynon-antigenic polymer, the present invention includes pharmaceuticallyacceptable salts of the compounds of formula (I). In addition, when R₃is ##STR5## (n) is preferably 0 (zero) or an integer ≧2.

In some preferred embodiments of the invention, R₃ includes both analpha and an omega linking group so that two equivalents of abiologically active ingredient or drug, designated herein as D and/orD', can be delivered. Each D (or D') is attached to the polymer via ahydrolyzable ester linkage. Thus, polymer-based mono- and bis-prodrugsare contemplated.

The prodrugs preferably include a water-soluble polyalkylene oxidepolymer as R₃. More preferably, R₃ is a polyethylene glycol and has amolecular weight of at least about 20,000.

In certain preferred aspects of the invention, the biologically activeor parent compound (designated D or D' herein) attached to the polymeris a taxane such as paclitaxel or taxotere. In other aspects of theinvention, the active or parent compound is camptothecin, etoposide,cis-platin derivatives containing OH groups, floxuridine orpodophyllotoxin. In still further embodiments, other oncolytic agents,non-oncolytic agents such as anti-inflammnatory agents, includingsteroidal compounds, as well as therapeutic low molecular weightpeptides such as insulin are also contemplated.

For purposes of the present invention, it will be understood that theterm "alkyl" shall be understood to include straight, branched orsubstituted C₁₋₁₂ alkyls, C₅₋₈ cycloalkyls or substituted cycloalkyls,etc.

One of the chief advantages of the compounds of the present invention isthat the prodrugs achieve a proper balance between the rate of parentdrug-polymer linkage hydrolysis and the rate of clearance of prodrugfrom the body. The linkage between the polymer and the parent compound,also referred to herein as a biologically-active nucleophile, hydrolyzesat a rate which allows a sufficient amount of the parent molecule to bereleased in vivo before clearance of the prodrug from the plasma orbody.

Another advantage of the present invention is that in certain preferredembodiments, the prodrug composition includes a racemic mixture of thelinker portion joining biologically active material linked to highmolecular weight polymers using both the (d) and (l) forms of theprodrug linkage. This unique blend allows the artisan to design a novelprodrug complex having controlled release properties in which there isan initial relatively rapid release of the drug from the prodrug form,due to the relatively rapid enzymatic cleavage of the (l) forms of theamino acid linker portion, followed by a relatively slow release of thedrug from the prodrug as a result of the hydrolysis of (d) form of theamino acid linker portion. Alternatively, the (d) and (l) forms of theamino acids can be used separately to employ the unique hydrolysisproperties of each isomer, i.e. (l)-relatively rapid, (d)-slowerhydrolysis. The compounds of the present invention are also designed toinclude polymers of adequate molecular weight to insure that thecirculating life of the prodrugs is sufficient to allow the necessaryamount of hydrolysis (and thus regeneration of therapeutic amounts ofthedrug in vivo) before elimination of the drug. Stated in another way, thecompounds of the present invention are preferably designed so that thecirculating life T_(1/2) is greater than the hydrolysis T_(1/2).

Methods of making and using the compositions described herein are alsoprovided.

BRIEF DESCRIPTON OF THE DRAWINGS

FIG. 1 is a schematic representation of the reactions carried out inaccordance with Examples 1-5.

FIG. 2 is a schematic representation of the reactions carried out inaccordance with Examples 6-17.

FIG. 3 is a schematic representation of the reaction carried out inaccordance with Example 18.

FIG. 4 is a schematic representation of the reaction carried out inaccordance with Example 19.

FIG. 5 is a schematic representation of the reactions carried out inaccordance with Example 20.

FIGS. 6 and 7 are schematic representations of the reactions carried outin accordance with Examples 21-28.

FIG. 8 is a schematic representation of the reaction carried out inaccordance with Example 29.

FIG. 9 is a schematic representation of the reaction carried out inaccordance with Example 30.

FIG. 10 is a schematic representation of the reaction carried out inaccordance with Example 31.

FIGS. 11 and 12 are schematic representations of the reactions carriedout in accordance with Examples 32-39.

FIG. 13 is a schematic representation of the reactions carried out inaccordance with Examples 12b and 15b.

FIG. 14 is a schematic representation of the reactions carried out inaccordance with Examples 21d and 22 IIb.

FIG. 15 is a schematic representation of the reactions carried out inaccordance with Example 22, Method A step IV.

FIG. 16 is a schematic representation of the reactions carried out inaccordance with Examples 29b and 29c.

FIG. 17, is a schematic representation of the reactions carried out inaccordance with Examples 47 through 53.

DETAILED DESCRIPTION OF THE INVENTION A. THE PRODRUGS

In most aspects of the invention, the prodrug compositions of thepresent invention contain hydrolyzable linkages between the polymerportion and a biologically active moiety derived from a biologicallyactive moiety or nucleophile, i.e. native or unmodified drug. Theselinkages are preferably ester linkages designed to hydrolyze at a ratewhich generates sufficient amounts of the biologically active parentcompound in a suitable time period so that therapeutic levels of theparent therapeutic moiety or moieties are delivered prior to excretionfrom or inactivation by the body. The term "sufficient amounts" forpurposes of the present invention shall mean an amount which achieves atherapeutic effect as such effect is understood by those of ordinaryskill in the art.

In one preferred embodiment of the invention, the prodrug compositionsof the invention comprise the formula set forth below: ##STR6## wherein:D is a residue of a biologically active moiety;

X is an electron withdrawing group;

Y and Y' are independently O or S;

(n) is zero (0) or a positive integer, preferably from 1 to about 12;

R₁ and R₂ are independently selected from the group consisting of H,C₁₋₆ alkyls, aryls, substituted aryls, aralkyls, heteroalkyls,substituted heteroalkyls and substituted C₁₋₆ alkyls; p1 R₃ is asubstantially non-antigenic polymer, C₁₋₁₂ straight or branched alkyl orsubstituted alkyl, C₅₋₈ cycloalkyl or substituted cycloalkyl,carboxyalkyl, carboalkoxy alkyl, dialkylaminoalkyl, phenylalkyl,phenylaryl or ##STR7## R₄ and R₅ are independently selected from thegroup consisting of H, C₁₋₆ alkyls, aryls, substituted aryls, aralkyls,heteroalkyls, substituted heteroalkyls, and substituted C₁₋₆ alkyls orjointly form a cyclic C₅ -C₇ ring.

Preferably, the polymer portion, designated R₃ herein, is furthersubstituted with a terninal capping moiety (Z) which is distal to theprimary linkage attaching D to the polymer. A non-limiting list ofsuitable capping groups includes OH, C₁₋₄ alkyl moieties, biologicallyactive and inactive moieties, or ##STR8## where D' is the same as D, adifferent biologically active moiety, a dialkyl urea, a C₁₋₄ alkyl,carboxylic acid or other capping group; and

Y, Y', R₁, R₂, X and (n) are as defined above.

Within Formula (I), Y and Y' are preferably oxygen and (n) is preferably1.

In preferred aspects of the invention, the linkage attaching D to thepolymer includes an amino acid ester spacer such as alanine. Thus, R₁and R₂ are preferably one of H, methyl or ethyl. Alternatively, when oneof R₁ and R₂ is H and the other is a substituted C₁₋₆ alkyl, it can beselected from carboxyalkyls, aminoalkyls, dialkylaminos, hydroxyalkyls,and mercaptoalkyls. In still further aspects of the invention, one of R₁and R₂ is H and the other is an aryl such as phenyl or an aralkyl suchas a benzyl or substituted benzyl.

In another embodiment, R₁ and R₂ are both not H when (n) is 1 (one) andR₃ is a polyalkylene oxide.

B. THE PRODRUG LINKAGE

1. The Electron Withdrawing Group X

Within the formula (I) described above, X is designated as an electronwithdrawing group. In particular, X can be selected from moieties suchas O, NR₁, S, SO and SO₂ where R₁ is as defined above. Preferably,however, when included as part of X, R₁ is H, a C₁₋₆ alkyl orsubstituted C₁₋₆₄ alkyl. For purposes of the present invention when (n)is 1 in Formulas (I) and (II), X is preferably a moiety which gives asubstituted acetic acid with a pKa of less than about 4.0 uponhydrolysis of the prodrug ester. The moieties selected for X within theformula promote relatively rapid hydrolysis because of the low pKa ofthe resulting substituted acetic acid.

On the other hand, when (n) is zero (0) and X is oxygen and Y and Y' areoxygen or sulfur, a carbonate or thiocarbonate linkage is formed. Thecarbonate linkage may also be cleaved by enzymes such as esteraseslocated in the plasma, as well as near or within the tumor area giventhe enhanced permeation and retention effect which occurs in solidtumors. See Greenwald, R. B. Exp. Opin. Ther. Patents (1997)7(6):601-609. Representative examples of carbonate derivatives aredescribed in Examples 47, 48, 49, 51a, 51b, 52, and 53.

Similarly, when (n) is zero (0), X is NR₁ and Y and Y' are oxygen orsulfur, a carbamate or thiocarbamate is formed. This linkage is morestable than carbonate or ester linkages but nonetheless may also becleaved by a variety of enzymes such as proteases and peptidases,largely in the tumor region. The drug is then targeted in the samemanner as carbamate or thiocarbamate linkages described above.Representative examples are described in Examples 50a and 50b.

2. The Amino Acid Portion of the Linker

As mentioned above in Section A, one aspect of the invention includesusing an amino acid ester spacer such as alanine within the linkageattaching the polymer R₂ to the biologically active moiety D. Thisportion of the linkage can be attached to the D portion directly asillustrated in FIG. 6 using t-Boc-l (or d or racemic)-alanine or byconverting a PEG acid or diacid with the l- or d-alanine-t-butyl esteras shown, for example, in FIG. 7.

3. Hydrolysis and Parent Drug Regeneration

The prodrug compounds of the present invention are designed so that inplasma the T_(1/2) circulation is greater than the T_(1/2) hydrolysis,which in turn is greater than the T_(1/2) for elimination, i.e.

    T.sub.1/2  circulation>T.sub.1/2  hydrolysis>T.sub.1/2  elimination.

The prior art had several shortcomings associated with its approach toproviding polymer based prodrugs. For example, in some cases, themolecular weight of the polymer was insufficient, i.e. 10,000 Daltons orless, regardless of the linkage used to attach the parent drug to thepolymer. In other cases, a polymer of sufficient molecular weight wasproposed but the linkage was not designed to allow sufficient in vivohydrolysis and release of the parent molecule. The compounds of thepresent invention overcome these shortcomings by including not onlypolymers of sufficient weight but also linkages which meet the criteriadiscussed above.

Regardless of whether (n) is 1, as preferred in the embodiment discussedabove in B1, the ester-based linkages included in the compounds have aT_(1/2) hydrolysis in the plasma of the mammal being treated which islong enough to allow the parent compounds to be released prior toelimination. Some preferred compounds of the present invention haveplasma T_(1/2) hydrolysis rates ranging from about 30 minutes to about12 hours. Preferably, the compositions have a plasma T_(1/2) hydrolysisranging from about 1 to about 8 hours and most preferably from about 2.5to about 5.5 hours. The compounds thus provide a distinct advantage overthe rapidly hydrolyzed prodrugs of the prior art, such as thosedescribed in U.S. Pat. No. 4,943,579 which are all about 45 minutes orless and are of limited practical value. The parent compounds appear tobe rapidly regenerated in vivo and quickly eliminated from circulation.While Applicants are not bound by theory, in those aspects of theinvention where prodrugs are formed, regeneration of sufficient amountsof the parent compound during the time the prodrug remains incirculation is believed to be a key to providing an effective prodrugcompositions.

C. SUBSTANTIALLY NON-ANTIGENIC POLYMERS

The prodrug compositions of the present invention include awater-soluble polymer, R₃. Suitable examples of such polymers includepolyalkylene oxides such as polyethylene glycols which are alsopreferably substantially non-antigenic. The general formula for PEG andits derivatives, i.e. R"--(CH₂ CH₂ O)_(x) --(CH₂)_(y) --R', where (x)represents the degree of polymerization or number of repeating units inthe polymer chain and is dependent on the molecular weight of thepolymer, (y) represents a positive integer, R' is (CHR₁) and R" is acapping group as defined herein or R'. It will be understood that thewater-soluble polymer will be functionalized for attachment to thelinkage designated X herein. As an example, the PEG can befunctionalized in the following non-limiting manner:

--C(Y)--(CH₂)_(n) --(CH₂ CH₂ O)_(x) --R",

--C(Y)--Y--(CH₂)_(n) --(CH₂ CH₂ O)_(x) --R",

--C(Y)--NR₁ --(CH₂)_(n) --(CH₂ CH₂ O)_(x) --R",

--CR₁ R₂ --(CH₂)_(n) --(CH₂ CH₂ O)_(x) --R".

where Y, R", R₁, R₂, (n) and (x) are as defined above.

In particular, polyethylene glycols (PEG's), mono-activated, C₁₋₄alkyl-terminated PAO's such as mono-methyl-terminated polyethyleneglycols (mPEG's) are preferred when mono-substituted polymers aredesired; bis-activated polyethylene oxides are preferred whendisubstituted prodrugs are desired. In order to provide the desiredhydrolyzable linkage, mono- or di-acid activated polymers such as PEGacids or PEG diacids are used. Suitable PAO acids can be synthesized byconverting mPEG--OH to an ethyl ester. See also Gehrhardt, H., et al.Polymer Bulletin 18: 487 (1987) and Veronese, F. M., et al., J.Controlled Release 10; 145 (1989). Alternatively, the PAO-acid can besynthesized by converting mPEG--OH into a t-butyl ester. See, forexample, commonly assigned U.S. patent application Ser. No. 08/440,732filed May 15,1995. The disclosures of each of the foregoing areincorporated by reference herein.

Although PAO's and PEG's can vary substantially in molecular weight,polymers having molecular weight ranges of at least 20,000 arepreferred. Polymers ranging from about 20,000 to about 80,000 areusually selected for the purposes of the present invention. Molecularweights of from about 25,000 to about 45, 000 are preferred and 30,000to about 42,000 are particularly preferred. The molecular weight of thepolymer selected for inclusion in the prodrug must be sufficient so asto provide sufficient circulation of the prodrug during hydrolysis ofthe linker.

The polymeric substances included herein are preferably water-soluble atroom temperature. A non-limiting list of such polymers includepolyalkylene oxide homopolymers such as polyethylene glycol (PEG) orpolypropylene glycols, polyoxyethylenated polyols, copolymers thereofand block copolymers thereof, provided that the water solubility of theblock copolymers is maintained.

As an alternative to PAO-based polymers, effectively non-antigenicmaterials such as dextran, polyvinyl alcohols, carbohydrate-basedpolymers, HPMA's and the like can be used if the same type of esteractivation is employed as described herein for PAO's such as PEG, i.e.conversion of alcohol to a 2-alkoxy acid. Those of ordinary skill in theart will realize that the foregoing list is merely illustrative and thatall polymeric materials having the qualities described herein arecontemplated. For purposes of the present invention, "effectivelynon-antigenic" means all polymeric materials understood in the art asbeing nontoxic and not eliciting an appreciable immune response inmammals.

As mentioned above, the prodrugs of the present invention include one ortwo equivalents of drug per equivalent of R₃, i.e. the polymer. As such,preferred polymers can be functionalized to form the bis-prodrugs whenreacted with a sufficient amount of a parent compound. One preferredpolymer-linkage combination is represented below as formula (III):##STR9## wherein: R₁₋₃, X and n are the same as that set forth above for(I);

Z¹ and Z² are independently CO₂ H, CO₂ R₆, OR₇, CONR₁ R₈, H, a C₁₋₄alkyl or ##STR10## where R₄ and R₅ are independently selected from thegroup consisting of H, C₁₋₆ alkyls, aryls, substituted aryls, aralkyls,heteroalkyls, substituted heteroalkyls, and substituted C₁₋₆ alkyls orjointly form a cyclic C₅ -C₇ ring;

R₆ is R₄ or an N-hydroxysuccinimide, an N-hydroxybenzotriazole, anN-acyl thazolidine, imidazole or an acid activating group;

R₇ is R₄ or C(O)-halogen, para nitrophenyl carbonate, imidazolylcarbonate, or N-hydroxysuccinimidyl carbonate; and

R₈ is R₄ or CR₁ R₂ CO₂ H.

In certain aspects of the invention, R₃ has a molecular weight of about20,000 or greater. Although, the prodrugs of the present invention canbe formed using any of the substantially non-antigenic polymersdescribed herein, the following polyalkylene oxide derivatives, i.e. PEGdiamines, PEG-acids and PEG-diacids are especially preferred for use information of the prodrug:

a) R₄ NR₅ --(CH₂)_(n) --O--PEG--O--(CH₂)_(n) --NR₄ R₅,

b) HO₂ C(CH₂)_(n) S--(CH₂)₂ --O--PEG--O--(CH₂)₂ --S--(CH₂)_(n) CO₂ H;

c) HO₂ C--CH₂ --O--PEG--O--CH₂ --CO₂ H; ##STR11## e) CH₃O--PEG--O--(CH₂)_(n) --CO₂ H; where R₁, R₂, and (n) are the same asdefined above for Formula (I).

It will be clear from the foregoing that other polyalkylene oxidederivatives of the foregoing, such as the polypropylene glycol acids,POG acids, etc., as well as other bifunctional linking groups are alsocontemplated.

D. PRODRUG CANDIDATES

1. Taxanes and Taxane Derivatives

One class of compounds included in the prodrug compositions of thepresent invention is taxanes. For purposes of the present invention, theterm "taxane" includes all compounds within the taxane family ofterpenes. Thus, taxol (paclitaxel), 3'-substitutedtert-butoxy-carbonyl-amine derivatives (taxoteres) and the like as wellas other analogs available from, for example, Sigma Chemical of St.Louis, Mo. are within the scope of the present invention. Representativetaxanes are shown below. ##STR12##

These compounds have been found to be effective anti-cancer agents.Numerous studies indicate that the agents have activity against severalmalignancies. To date, their use has been severely limited by, amongother things, their short supply, poor water solubility andhypersensitivity. It is to be understood that other taxanes includingthe 7-aryl-carbamates and 7-carbazates disclosed in commonly assignedU.S. Pat. Nos. 5,622,986 and 5,547,981 can also be included in theprodrugs of the present invention. The contents of the foregoing U.S.patents are incorporated herein by reference.

Although the examples describe inter alia paclitaxel for illustrativepurposes, it is to be understood that the methods described herein aresuitable for all taxanes and related molecules. The only limitation onthis provision is that the selected taxanes must be capable ofundergoing 2' position modifications described herein. Paclitaxel,however, is a preferred taxane.

Synthesis of the taxane-based prodrugs of the invention is set forthbelow in section E and in the Examples. In general, however, a taxanehaving the 2'-position available for substitution is reacted with asuitably activated polymer such as a PEG acid under conditionssufficient to cause the formation of a 2' ester linkage between the twosubstituents. The corresponding diester can be prepared by reacting atleast about 2 equivalents of taxane per polymer diacid. Even when twoequivalents of taxane are reacted with the polymer diacid, the resultingconjugate can contain minor amounts (i.e. up to 25%) by weight of amonoester species containing an acyl urea or carboxylic acid distal tothe polymer-taxane linkage with regard to the polymer. Thesecompositions are also capable of delivering a biological effect. It ispreferred that the polymer acid have a molecular weight of at leastabout 20,000. See FIGS. 5 and 8 as illustrative examples.

2. Camptothecin and Related Topoisomerase I Inhibitors

Camptothecin is a water-insoluble cytotoxic alkaloid produced bycamptoteca accuminata trees indigenous to China and nothapodyes foetidatrees indigenous to India. Camptothecin and related compounds andanalogs are also known to be potential anticancer or antitumor agentsand have been shown to exhibit these activities in vitro and in vivo.Camptothecin and related compounds are also candidates for conversion tothe prodrugs of the present invention. See, for example, U.S. Pat. No.5,004,758 and Hawkins, Oncology, December 1992, pages 17-23.Camptothecin and related analogues have the structure:

    __________________________________________________________________________                                              (V)                                  ##STR13##                                                                    20(S)-Camptothecin                                                                       Position                                                           Camptothecin analogues                                                                   7      9     10                                                    __________________________________________________________________________    Topotecan  H      CH.sub.2 N(CH.sub.3).sub.2                                                          OH                                                    CPT-11     --CH.sub.2 --CH.sub.3                                                                H                                                                                    ##STR14##                                            __________________________________________________________________________

Additional camptothecin analogs include those reported in the literatureincluding the 10-hydroxycamptothecins, 11-hydroxycamptothecins and/or10,11-dihydroxycamptothecins, 7- and/or 9-alkyl, substituted alkyl,cycloalkyl, alkoxy, alkenyl, aminoalkyl, etc. camptothecins, A-ringsubstituted camptothecins such as 10,11-alkylenedioxycamptothecins, suchas those disclosed in U.S. Pat. No. 5,646,159, the contents of which areincorporated herein by reference, etc.

Formation of a monoester camptothecin prodrug can be accomplished byreacting one or more equivalents of a suitably (acid) activated polymerwith one equivalent of the camptothecin derivative under conditionssufficient to effectively convert the 20-OH to an ester-linked polymericbased prodrug. Camptothecin diesters are similarly prepared by reactingat least about 2 and preferably greater equivalents of the camptothecinwith a suitably prepared PAO diacid. Details concerning the reactionschemes and conditions are provided in Section E, below, FIGS. 1, 2, 4,etc. and in the Examples.

In addition to the foregoing camptothecin analogs, it has been foundthat new 20(S)camptothecin-mono-PEG ester compounds can be formed when adiacid PEG is used with certain carbodiimide condensing agents with theappropriate stoichiometry. For example, the alpha terminus of thepolymer is converted to a camptothecin-PEG ester and the omega terminusof the PEG diacid is converted from the acid to an acyl dialkyl urea,depending on the dialkyl carbodiimide employed to effect conjugation.These derivatives show antitumor activity in vivo and upon NMRinspection, cross-linking was found to be negligible. In most preferredaspects, however, bis-prodrug camptothecin compositions are formed bylinking each of the alpha and omega termini of the polymer via Y' to the20 S position of camptothecin when a carbodiimide is used as thecondensing agent. In alternative aspects, higher amounts of the diestercan be obtained by the use of a Mukaiyama reagent, i.e.2-chloro-1-methylpyridinium iodide.

3. Additional Biologically-Active Moieties

In addition to the foregoing molecules, the prodrug formulations of thepresent invention can be prepared using many other compounds. Forexample, biologically-active compounds such as cis-platin derivativescontaining OH groups, i.e. ##STR15## mono- and bis-PEG esters derivedfrom floxuridine, shown below: ##STR16## podophyllotoxin, shown below:##STR17## and related compounds can be included. The prodrug ester canbe formed at the 2-hydroxy position for the "A" cis-platin derivative,the 2-hydroxyethyl position of the "B" cis-platin derivative, the 3' and5' hydroxy positions of floxuridine and at the C-4 hydroxy forpodophyllotoxin. These prodrug compositions can be prepared, forexample, using the technique described for preparing compound 32 inExample 31.

The parent compounds selected for prodrug forms need not besubstantially water-insoluble, although the polymer-based prodrugs ofthe present invention are especially well suited for delivering suchwater-insoluble compounds. Other useful parent compounds include, forexample, certain low molecular weight biologically active proteins,enzymes and peptides, including peptido glycans, as well as otheranti-tumor agents, cardiovascular agents such as forskolin,anti-neoplastics such as combretastatin, vinblastine, vincristine,doxorubicin, AraC, maytansine, etc. anti-infectives such as vancomycin,erythromycin, etc. anti-fungals such as nystatin or amphoteracin B,anti-anxiety agents, gastrointestinal agents, central nervoussystem-activating agents, analgesics, fertility or contraceptive agents,anti-inflammatory agents, steroidal agents, anti-urecemic agents,cardiovascular agents, vasodilating agents, vasoconstricting agents andthe like.

The foregoing is illustrative of the biologically active moieties whichare suitable for the prodrugs of the present invention. It is to beunderstood that those biologically active materials not specificallymentioned but having suitable ester-forming groups, i.e. hydroxylmoieties, are also intended and are within the scope of the presentinvention. It is also to be understood that the prodrug conjugates ofthe present invention may also include compounds containing not only oneequivalent of drug and polymer but also a moiety which does not effectbioactivity in vivo. For example, it has been found that in someinstances, in spite of reacting diacids with drug molecules having asingle linkage point, the reaction conditions do not provide prodrugswith two equivalents of drug per polymer. On the contrary, the prodrugscontain only one equivalent of drug per polymer. By-products of thereactants such as acyl ureas can be formed. Furthermore, it has alsobeen found that in spite of the reaction with a bis-activated polymer,the prodrugs are remarkably free of cross-linked species.

The only limitation on the types of molecules suitable for inclusionherein is that there is at least one position on which the hydrolyzablelinkage can be attached, so that after prodrug administration, theprodrug can regenerate sufficient quantities of the parent compound invivo.

E. SYNTHESIS OF PRODRUGS

Generally, the prodrugs of the invention are prepared by:

1) providing an activated polymer, such as a PEG-acid or PEG-diacid anda parent compound having a position thereon which will allow ahydrolyzable linkage to form, and

2) reacting the two substituents in an inert solvent such as methylenechloride, chloroform, toluene or DMF in the presence of a couplingreagent such as 1,3-diisopropylcarbodiimide (DIPC), 1, (3-dimethylaminopropyl) 3-ethyl carbodimide (EDC), any suitable dialkylcarbodiimide, Mukaiyama reagents, (e.g. 2-halo-1-alkyl-pyridiniumhalides) or propane phosphonic acid cyclic anhydride (PPACA), etc. whichare available, for example from commercial sources such as SigmaChemical, or synthesized using known techniques and a base such asdimethylaminopyridine (preferred), diisopropyl ethylamine, pyridine,triethylamine, etc. at a temperature from 0° C. up to 22° C. (roomtemperature).

In another preferred aspect of this embodiment, the synthesis methodprovides polymer-based prodrugs having a circulation half-life greaterthan their in-vivo hydrolysis half-life. The method includes:

reacting a biologically active moiety containing an available hydroxylgroup with a bifunctional spacer moiety containing an availablecarboxylic acid group in the presence of a first coupling agent to forma biologically active moiety--spacer prodrug intermediate,

reacting the biologically active moiety--spacer prodrug intermediatewith a substantially non-antigenic polymer containing a terminalcarboxylic acid group or a terminal amine or hydroxy group in thepresence of a second coupling agent and recovering the polymer-basedprodrug.

The first and second coupling agents can be the same or different.

Examples of suitable bifunctional spacer groups include diglycolic acid,thiodiglycolic acid, l-alanine and d-alanine, hydroxyacetic acid,bromoacetic acid, etc.

An illustrative example of method of preparing the conjugates usingcamptothecin derivatives as the prototypical biologically activenucleophile includes the steps of:

forming a camptothecin derivative containing bifunctional spacercontaining moiety, by contacting the camptothecin derivative with abifunctional spacer containing moiety such as tBoc, d or l alanine inthe presence of a coupling agent such as DIPC or PPAC;

forming the trihaloacetic acid deriviative of the camptothecinderivative containing the bifunctional spacer containing moiety such asthe trifluoroacetic acid salt; and

reacting the trihaloacetic acid derivative of the camptothecinderivative containing the bifunctional spacer containing moiety with adiacid derivative of a substantially non-antigenic polymer such as a PEGdiacid. The resultant compound is recovered using known techniques.

Alternative and specific syntheses are provided in the examples. Oneparticular alternative, however, includes derivatizing the biologicallyactive moiety in the position desired for the linkage and thereafterreacting the derivative with an activated polymer.

F. NON-POLYMERIC DERIVATIVES

In certain other aspects of the invention, the compounds of formula (I)are non-polymeric derivatives, i.e. R₃ is other than a substantiallynon-antigenic polymer. R₃, instead is a C₁₋₁₂ straight or branched alkylor substituted alkyl, C₅₋₈ cycloalkyl or substituted cycloalkyl,carboxyalkyl, carboalkoxy alky, dialkylaminoalkyl, phenylalkyl,phenylaryl or ##STR18## R₄ and R₅ are independently selected from thegroup consisting of H, C₁₋₆ alkyls, aryls, substituted aryls, aralkyls,heteroalkyls, substituted heteroalkyls, and substituted C₁₋₆ alkyls orjointly form a cyclic C₅ -C₇ ring. It is preferred that when R₃##STR19## (n) is preferably 0 (zero) or an integer ≧2.

The compounds of this aspect of the invention are thus simple carbonate,simple carbamate derivatives and the like and can have utility eithertherapeutically as is or as pharmaceutically acceptable salts such asthe sodium salt. Preparation of such salts will be apparent to those ofordinary skill without undue experimentation. These compounds are alsouseful as intermediates which can be further processed into prodrugs.For example, when R₃ is a substituted alkyl or cycloalkyl, substitutedaryl or heteroaryl, or substituted aralkyl, these intermediate compoundscan be reacted with activated polymers as described herein to formprodrugs. In particular, if R₃ is an aminoalkyl such as aminopropyl,aminobutyl, aminoethyl, etc., the intermediate can be reacted withnon-antigenic polymer containing a succinimidyl carbonate activated,cyclic imide thione activated polymer or with an acid derivative of thepolymer using a condensing agent. Similarly, if R₃ is a carboxyalkylsuch as a carboxypropyl, carboxybutyl, carboxyethyl, etc., theintermediate is reacted with an amine-activated substantiallynon-antigenic polymer such as a PEG-amine. Alternatively, if R₃ is ahydroxyalkyl such as a hydroxypropyl, hydroxybutyl, hydroxyethyl, etc.,the intermediate is reacted with an isocyanate or isothiocyanteactivated substantially non-antigenic polymer such as a PEG--NCO orPEG--NCS. Simple amino acid esters of the non-polymeric derivatives ofthe present invention are also contemplated.

G. METHODS OF TREATMENT

Another aspect of the present invention provides methods of treatmentfor various medical conditions in mammals. The methods includeadministering to the mammal in need of such treatment, an effectiveamount of a prodrug, such as a paclitaxel 2'-PEG ester, which has beenprepared as described herein. The compositions are useful for, amongother things, treating neoplastic disease, reducing tumor burden,preventing metastasis of neoplasms and preventing recurrences oftumor/neoplastic growths in mammals.

The amount of the prodrug administered will depend upon the parentmolecule included therein. Generally, the amount of prodrug used in thetreatment methods is that amount which effectively achieves the desiredtherapeutic result in mammals. Naturally, the dosages of the variousprodrug compounds will vary somewhat depending upon the parent compound,rate of in vivo hydrolysis, molecular weight of the polymer, etc. Ingeneral, however, prodrug taxanes are administered in amounts rangingfrom about 5 to about 500 mg/m² per day, based on the amount of thetaxane moiety. Camptothecin and podophyllotoxin prodrugs are alsoadministered in amounts ranging from about 5 to about 500 mg/m² per day.The range set forth above is illustrative and those skilled in the artwill determine the optimal dosing of the prodrug selected based onclinical experience and the treatment indication.

The prodrugs of the present invention can be included in one or moresuitable pharmaceutical compositions for administration to mammals. Thepharmaceutical compositions may be in the form of a solution,suspension, tablet, capsule or the like, prepared according to methodswell known in the art. It is also contemplated that administration ofsuch compositions may be by the oral and/or parenteral routes dependingupon the needs of the artisan. In preferred aspects of the invention,however, the prodrugs are parenterally administered to mammals in needthereof.

H. EXAMPLES

The following examples serve to provide further appreciation of theinvention but are not meant in any way to restrict the effective scopeof the invention. The numbers shown in bold in parentheses in theExamples correspond to the compounds shown in the schematic diagrams setforth in the Figures.

Example 1

Camptothecin-20-O-ester of Benzyloxyacetic Acid--Compound 2:

Referring now to FIG. 1, benzyloxyacetic acid 1 (2 g, 12.06 mmol),prepared by the aqueous hydrolysis of benzyloxyacetyl chloride,(Aldrich) diisopropylcarbodiimide (DIPC, 1.9 ml, 12.06 mmol) anddimethylaminopyridine (DMAP, 982 mg, 8.04 mmol) were added to asuspension of camptothecin, (1.4 g, 4.02 mmol) in CH₂ Cl₂ (500 ml) at 0°C. Stirring was continued for 3 hours. The resulting yellow solution wasconcentrated to about 100 ml and washed with 1N hydrochloric acid (10ml×2), followed by 1% aqueous sodium bicarbonate solution (10 ml×2). Theorganic layer was dried (anhydrous MgSO₄) and evaporated in vacuo togive a yellow solid which was recrystallized from ethyl acetate. Theproduct was then triturated with methanol (10 ml), and the slurryfiltered to yield 2 (1.3 g, 65%).

¹ H NMR(CDCl₃) δ: 1.0(t), 1.84(s), 2.1-2.3(m), 4.31(s), 4.59-4.69(q),5.28(s), 5.4-5.8(dd), 7.22(s), 7.27(s), 7.3-7.38(m), 7.6-7.7(m),7.81-7.87(m), 7.92-7.95(d), 8.19-8.22(d), 8.39(s). ¹³ C NMR(CDCl₃) δ:7.52, 31.74, 49.90, 66.59, 67.16, 73.27, 76.38, 95.80, 120.27, 127.97,128.10, 128.43, 129.54, 130.63, 131.15, 136.81, 145.39, 146.36, 148.81,152.22, 157.26, 167.19, 169.52.

Example 2

Camptothecin-20-O-ester of Hydroiyacetic Acid--Compound 3:

Continuing with reference to FIG. 1, a suspension of 2 (1 g, 2.01 mmol)and 10% Pd/C (500 mg) in ethanol (100 mL) was degassed by sparging withnitrogen, followed by the addition of cyclohexene (5 mL). The reactionmixture was heated at reflux for 20 hours and the catalyst was filtered.Removal of solvent in vacuo followed by recrystallization fromacetonitrile gave 3 (500 mg, 50%).

¹ H NMR (DMSO-D6) δ: 1.0(t), 1.84(s), 2.1-2.3(m), 3.1(s), 4.31(s),4.0-4.69(m), 5.28(s), 5.6(m), 5.8(m), 7.22(s), 7.6-7.7(m), 7.6-7.95(m),8.0-8.2(m), 8.3(s), 8.7(s). ¹³ C NMR (DMSO-D6) δ 7.5, 30.14, 50.20,59.37, 66.21, 75.83, 79.14, 94.95, 118.84, 127.71, 127.97, 128.52,128.87, 129.77, 130.42, 131.58, 145.35, 145.95, 147.85, 152.29, 156.53,167.21, 171.69.

Example 3

Camptothecin-20-O-ester of 2-Imidazolyl Carbonyloxyacetic Acid--Compound4:

As schematically shown in FIG. 1, a solution of 3 (240 mg, 0.59 mmol),N,N-carbonyldiimidazole (288 mg, 1.77 mmol) in chloroform (80 mL) wasstirred at 50° C. for 18 hours. Removal of solvent in vacuo followed bytrituration with ethyl acetate gave 4 as a pale yellow solid (170 mg,58%).

¹ H NMR (CDCl₃) δ: 1.0(t), 2.1-2.5(m), 5.1(d), 5.28(s), 5.4-5.8(dd),7.0(s), 7.5(s), 7.7(m), 7.9(m), 8(d), 8.1(s), 8.3(d), 8.4(s).

Example 4

Camptothecin-20-O-ester of 2-PEG_(40kDa) Carbamoylacetic Acid--Compound5a:

Method A:

As shown in FIG. 1, a solution of 3 (60.4 mg, 0.148 mmol), PEG_(40k)diisocyanate (2 g, 0.05 mmol), dibutyl tin dilaurate (15.7 mg, 0.024mmol) in dichloromethane (20 mL) was refluxed for 18 hours. Removal ofthe solvent in vacuo and recrystallization from 2-propanol (10 mL) gave5a.

Example 5

Camptothecin-20-O-ester of 2-PEG_(40kDa) Carbamoylacetic Acid--Compound5a:

Method B:

As illustrated in FIG. 1, solution of 4 (74 mg, 0.148 mmol) andPEG_(40kDa) diamine (12, 2 g, 0.05 mmol) in 2-propanol (20 mL) wasrefluxed for 12 hours. The solvent was removed under reduced pressure toyield 5a as a solid which was recrystallized from 2-propanol (1.6 g,79%).

Example 6

a) Camptothecin-20-O-ester of 2-PEG_(40k) N-methylcarbamoyl AceticAcid--Compound 5b:

FIG. 1 also shows formation of compound 5b. Compound 5b was prepared ina similar manner as that used to prepare compound 5a in Example 5(method B), using PEG_(40k) N-methyldiamine 13 as a starting material.

¹³ C NMR (CDCl₃) δ: 166.63, 166.08, 156.17, 151.39, 147.84, 145.41,144.35, 130.84, 130.03, 128.95, 127.99, 127.71, 127.61, 127.40, 118.68,94.30, 76.72, 65.66-71.65(PEG), 61.3, 49.37, 48.52, 48.15, 47.82, 35.14,34.54, 33.15, 30.97, 21.62, 6.97.

b) PEG_(40k) Dicarboxylic Acid Ester of 20-O-hydroxyacetylCamptothecin--Compound 5c:

FIG. 1 also illustrates formation of compound 5c. PEG 40_(kDa)dicarboxylic acid (3 grams, 0.075 mmol) was azeotroped in toluene (100ml) for 1 hour. Potassium t-butoxide, 1.0 M solution (165 μl, 0.165mmol) was added to the solution and the contents were refluxed for 2hours. The solvent was removed under reduced pressure and the solidobtained was redissolved in DMSO (30 ml). Compound 3 (140 mg, 0.299mmol) was added to this solution and the reaction mixture was stirred atroom temperature for 18 hours. Ether (100 ml) was added and the solidprecipitated was collected by filtration and recrystallized from2-propanol to yield 5c.

Example 7

Mono t-butyl Ester of Diglycolic Acid 6:

Referring now to FIG. 2, a solution of diglycolic anhydride (10 g, 0.09mol) and DMAP (10.5 g, 0.09 mol) in dry t-butanol (75 ml) was stirred atreflux temperature for 18 hours. The solvent was removed under reducedpressure and the residue was dissolved in water (100 ml). The aqueoussolution was acidified to pH 2.5 to 3.0 with 1N HCl and extracted withdichloromethane. Removal of solvent from the dried extracts yielded 12.3g (75%) of the mono t-butyl ester of glycolic acid 6. ¹³ C NMR (CDCl₃)δ: 172.85, 169.54, 82.42, 68.78, 68.45, 27.84.

Example 8

Camptothecin-20-O-ester of 6--Compound 8:

Continuing with reference to FIG. 2, a mixture of 6, (4.2 g, 0.02 mol),camptothecin (4.0 g, 0.01 mol), dimethylamino pyridine (DMAP, 2.7 g,0.02 mol), and diisopropylcarbodiimide DIPC 2.8 g, 0.02 mol) inanhydrous dichloromethane (40 ml) was stirred for 18 hours at roomtemperature. The reaction mixture was washed with water, then saturatedaqueous sodium bicarbonate, 0.1N HCl and again with water. The organiclayer was dried (anhyd.MgSO₄) and the solvent removed in vacuo.Recrystallization of the resultant solid from dichloromethane/ether gave8, (3.1 g, 54%).

¹ H NMR (CDCl₃) δ: 8.35(s), 8.15-8.18(d), 7.89-7.92(d); 7.80(m),7.63-7.66(m), 7.20(s), 5.35-5.72 (ABq), 5.21(s), 4.45-4.48(d),4.11-4.13(d), 2.2-2.3(m), 1.45(s), 1.00(t).

¹³ CNMR (CDCl₃) δ: 168.97, 168.52, 166.98, 157, 151.94, 148.55, 146.25,145.13, 130.97, 130.39, 129.32, 128.21, 127.99, 127.76, 119.94, 95.54,81.75, 76.38, 68.36, 67.45, 66.96, 49.72, 31.53, 27.86, 7.38.

Example 9

Camptothecin-20-O-ester of Diglycolic Acid 10:

Continuing with reference to FIG. 2, compound 8 (0.8 g, 1.5 mmol) indichloromethane-trifluroacetic acid solution (12 ml, 8:4) was stirred atroom temperature for 30 minutes. The solvent was removed under reducedpressure, and the resulting solid was recrystallized fromdichloromethane-ether to yield 10 (0.6 g, 82%).

The final product 10 can also be made directly by condensing 20(S)camptothecin with diglycolic anhydride in methylene chloride and thepresence of DMAP.

¹ H NMR (CDCl₃) δ: 8.44(s), 8.27-8.34(m), 7.95(d), 7.86(m)7.70(m),7.34(s), 5.37-5.75 (ABq,) 5.31(s), 4.47-4.5(d),4.39-4.41(d)2.18-2.26(m), 1.03(t).¹³ C NMR (DMSO-D₆) δ: 171.15, 169.27,167.41, 156.84, 152.63, 148.19, 146.41, 145.45, 131.88, 130.71, 130.11,129.22, 128.83, 128.29, 128.02, 119.08, 95.24, 76.72, 67.57, 67.33,66.62, 50.54, 30.47, 7.84.

Example 10

PEG_(40kDa) di-N-methylamine Hydrochloride 12:

Continuing with reference to FIG. 2, PEG_(40kDa) dichloride was preparedby using a procedure similar to that reported for synthesizing mPEG_(5k)Cl, Greenwald et al. J. Org. Chem., 1995, 60, 331-336, the contents ofwhich are incorporated herein by reference. A solution of thePEG_(40kDa) dichloride in 40% methylamine (400 ml) was then placed in asealed polypropylene bottle and heated at 60° C. for 3 days. Subsequentremoval of the solvent from the reaction mixture followed byrecrystallization from 2-propanol (1.5 L) yielded 12 (44 g, 87%). ¹³ CNMR (CDCl₃) δ: 33.10, 48.38, 66.18-71.60 (PEG).

Example 11

PEG_(40kDa) Amide of Acid 10--Compound 14:

Continuing with reference to FIG. 2, PEG_(40kDa) diamine hydrochloride13 was prepared following a procedure similar to that reported formPEG_(5k) NH₂ by Greenwald et al. J. Org. Chem., 1995, 60, 331-336, (thecontents of which were incorporated by reference supra). A mixture of10, (0.14 g, 0.3 mmol), the PEG_(40kDa) diamine hydrochloride 13, (3.0g, 0.075 mmol), DMAP (55 mg, 0.45 mmol), and DIPC (38 mg, 0.3 mmol) inanhydrous dichloromethane (30 ml) was stirred for 18 hours at roomtemperature. Removal of the solvent in vacuo and recrystallization from2-propanol gave 14 (2.8 g, 90%).

¹³ C NMR (CDCl₃) δ: 168.39, 168.09, 166.51, 156.8, 151.80, 148.44,146.22, 144.76, 131.04, 130.32, 129.17, 128.17, 127.91, 127.71, 119.77,95.05, 76.72, 66.73-71.89 (PEG), 49.64, 38.24, 31.38, 7.17.

Example 12

a) PEG_(40kDa) N-methylamide of Acid 10--Compound 15:

As shown in FIG. 2, a mixture of 10, (0.14 g, 0.3 mmol), PEG_(40kDa)di-N-methylamine hydrochloride 13, (3.0 g, 0.075 mmol), DMAP (55 mg,0.45 mmol), and DIPC (38 mg, 0.3 mmol) in anhydrous dichloromethane (30ml) was stirred for 18 hours at room temperature. Removal of the solventin vacuo and recrystallization from 2-propanol gave 15 (2.8 g, 90%).

¹³ C NMR (CDCl₃) δ: 168.73, 168.09, 166.51, 156.58, 151.80, 148.18,145.91, 144.77, 130.84, 130.03, 128.95, 127.99, 127.71, 127.61, 127.40,119.77, 95.05, 76.72, 66.46-71.65 (PEG), 49.41, 48.3, 47.5, 35.24, 33.3,31.06, 6.97.

b) PEG_(40kDa) Ester of Acid 10--Compound 44:

Referring now to FIG. 13, a mixture of 10, (0.14 g, 0.3 mmol),PEG_(40kDa) dicarboxylic acid 43, see Example 22 infra, (3.0 g, 0.075mmol), DMAP (55 mg, 0.45 mmol), and DIPC (38 mg, 0.3 mmol) in anhydrousdichloromethane (30 ml) was stirred for 18 hours at room temperature.Removal of the solvent in vacuo and recrystallization from 2-propanolgave 44 (2.8 g, 90%).

Example 13

Mono t-Butyl Ester of Thiodiglycolic Acid--Compound 7:

As shown in FIG. 2, a solution of thiodiglycolic anhydride (10 g, 0.09mol) and DMAP (10.5 g, 0.09 mol) in t-butanol (75 ml) was stirred atreflux temperature for 18 hours. The solvent was removed under reducedpressure and the residue dissolved in water (100 ml). The aqueoussolution was acidified to pH 2.5 to 3.0 with 1N HCl and extracted withdichloromethane. Removal of the solvents from the dried extracts yieldedthe mono t-butyl ester of thiodiglycolic acid 7. ¹³ C NMR (CDCl₃) δ:174.55, 168.95, 81.83, 34.43, 33.00, 27.49, 21.23.

Example 14

Camptothecin-20-O Ester of 7--Compound 9:

Continuing with reference to FIG. 2, a mixture of 7, (4.2 g, 0.02 mol),camptothecin (4.0 g, 0.01 mol), DMAP (2.7 g, 0.02 mol), and DIPC (2.8 g,0.02 mol) in anhydrous dichloromethane (40 ml) was stirred for 18 hoursat room temperature. The reaction mixture was then washed with water,saturated aqueous sodium bicarbonate, 0.1N HCl and finally with water.The organic layer was dried (anhyd.MgSO₄) and removal of the solvent invacuo and recrystallization of the resultant residue fromdichloromethane/ether gave 9.

¹³ C NMR (CDCl₃) δ: 168.81, 168.65, 167.10, 157.33, 152.25, 148.86,146.35, 145.63, 130.84, 131.13, 130.62, 129.66, 128.41, 128.15, 128.02,120.11, 95.86, 81.96, 76.53, 67.03, 49.95, 34.27, 32.87, 31.67, 27.94,7.57.

Example 15

Camptothecin-20-O-mono Ester of Thiodiglycolic Acid--Compound 11:

As shown in FIG. 2, compound 9 (0.8 g, 1.5 mmol) indichloromethane-trifluroacetic acid solution (12 ml, 8:4) was stirred atroom temperature for 30 minutes. The solvent was removed under reducedpressure, and the resulting solid was recrystallized fromdichloromethane/ether to yield 11.

Example 16

a) PEG_(40kDa) Amide of Acid 11--Compound 16:

A mixture of 11, (0.14 g, 0.3 mmol), PEG_(40kDa) diamine hydrochloride13, (3.0 g, 0.075 mmol), DMAP (55 mg, 0.45 mmol), and DIPC (38 mg, 0.3mmol) in anhydrous dichloromethane (30 ml) was stirred for 18 hours atroom temperature. Removal of the solvent in vacuo and re-crystallizationof the solid product was accomplished from 2-propanol to give 16. SeeFIG. 2.

b) PEG_(40kDa) Ester of Acid 11--Compound 45:

Referring now to FIG. 13, a mixture of 11, (0.14 g, 0.3 mmol),PEG_(40kDa) diol, see Example 22 infra, (3.0 g, 0.075 mmol), DMAP (55mg, 0.45 mmol), and DIPC (38 mg, 0.3 mmol) in anhydrous dichloromethane(30 ml) was stirred for 18 hours at room temperature. Removal of thesolvent in vacuo and recrystallization of the solid product wasaccomplished from 2-propanol to give 45.

Example 17

PEG_(40kDa) N-methylamide of Acid 11--Compound 17:

A mixture of 11, (0.14 g, 0.3 mmol), PEG_(40kDa) di-N-methylaminehydrochloride 12, (3.0 g, 0.075 mmol), DMAP (55 mg, 0.45 mmol), and DIPC(38 mg, 0.3 mmol) in anhydrous dichloromethane (30 ml) was stirred for18 hours at room temperature. Removal of the solvent in vacuo andrecrystallization from 2-propanol gave 17. See FIG. 2.

Example 18

PEG_(40kDa) Thioacetic Acid 18:

Referring now to FIG. 3, a mixture of of PEG_(40kDa) dichloride, (40.0g, 1 mmol), ref. Greenwald et al. J. Org. Chem., 1995, 60, 331-336),thioacetic acid (3.6 g, 40 mmol) and sodium hydroxide (4.0 g, 40 mmol)in water (160 ml) was placed in a sealed polyethylene bottle and placedin a water bath at 70° C. for 18 hours. The reaction mixture was cooledto room temperature, diluted with 100 ml of water and acidified withhydrochloric acid to pH 2.0, followed by extraction with methylenechloride. The combined extracts were dried over magnesium sulfate,filtered, and the solvent removed by rotary evaporator. The solidresidue was recrystallized from 2-propanol to yield 35 g (69%) ofproduct. ¹³ C NMR (CDCl₃) δ: 31.17, 33.23, 58.24, 170.66.

Example 19

Camptothecin-20-O-ester of 18--Compound 19:

Referring now to FIG. 4, PEG_(40kDa) thioacetic acid (18, 16 g, 0.40mmol) was dissolved in 250 mL of anhydrous methylene chloride at roomtemperature. To this solution, DIPC (280 mg, 2.2 mmol), DMAP (280 mg,2.2 mmol) and camptothecin (800 mg, 2.2 mmol) were added at 0° C. Thereaction mixture was allowed to warm to room temperature and left for 16hours followed by removal of the solvent in vacuo. The residue wasrecrystallized from 2-propanol to yield 13.6 g, 77%. The product wasfound to be a mixture of 19a+19b.

Example 20

Paclitaxel-2'-O-ester of 18--Compound 20:

Referring now to FIG. 5, PEG_(40kDa) thioacetic acid (18, 8 g, 0.20mmol) was dissolved in 120 mL of anhydrous methylene chloride at roomtemperature. To this solution, DIPC (108 μL, 0.70 mmol), DMAP (86 mg,0.70 mmol) and paclitaxel (606 mg, 0.70 mmol) were added at 0° C. Thereaction mixture was allowed to warm to room temperature and left for 16hours. The solution was washed with 0.1N HCl, dried and evaporated underreduced pressure to yield a white solid (15 g, 80%) which wasrecrystallized from 2-propanol. The product was found to be a mixture of20a+20b.

¹³ C NMR (CDCl₃) δ: 8.59, 13.60, 19.62, 19.70, 20.92, 21.0, 21.29,21.65, 25.66, 30.34, 30.72, 31.92, 34.82, 41.86, 42.13, 44.74, 44.81,47.32, 52.07, 57.15, 66.36-71.13(PEG), 73.66, 73.95, 74.36, 74.4, 79.89,83.23, 126.19, 126.43, 126.77, 126.82, 127.42, 127.68, 127.79, 127.96,128.59, 129.04, 129.22, 130.68, 131.95, 132.51, 132.89, 136.05, 140.99,152.57, 165.29, 166.29, 166.98, 168.73, 169.44, 202.33.

Example 21

a) Camptothecin-20-O-(l)Alanate TFA Salt (23):

Referring now to FIG. 6, tBoc-l-Alanine (1.8 g, 9.39 mmol) was dissolvedin 700 mL of anhydrous methylene chloride at room temperature. To thissolution, DIPC (1.5 ml 9.39 mmol), DMAP (765 mg, 6.26 mmol) andcamptothecin (1.09 g, 3.13 mmol) were added at 0° C. The reactionmixture was allowed to warm to room temperature and left for 16 hours.The solution was washed with 0.1N HCl, dried and evaporated underreduced pressure to yield a white solid which was recrystallized frommethanol to give Camptothecin-20-O-ester of t-Boc-l-Alanine 21. ¹ HNMR(DMSO-D₆): δ 0.9(t), 1.3(d). 1.6(s), 2.1(m), 4(m), 5.3(s), 5.5(s),7.3(s), 7.5-8.8(m).

b) Compound 21 (1.19 g, 2.12 mmol) was dissolved in a mixture ofmethylene chloride (15 ml) and trifluoroacetic acid (15 ml) and stirredat room temperature for 1 hour. The solvent was removed and the solidwas recrystallized from methylene chloride and ether to give (1 g) ofproduct 23 as the TFA salt.

¹ H NMR(DMSO-D₆) δ: 1.0(t), 1.6(d), 2.2(m), 4.4(m), 5.4(s), 5.6(s),7.2(s), 7.7-8.8(m).

¹³ C NMR (DMSO-D₆) δ: 7.5, 15.77, 30.09, 47.8, 50.27, 66.44, 77.5,94.92, 119.10, 127.82, 128.03, 128.62, 128.84, 129.75, 130.55, 131.75,144.27, 146.18, 147.90, 152.24, 156.45, 166.68, 168.69.

c) Camptothecin-20-O-(d/l) Alanate TFA Salts:

The d-alanate and d/l racemic alanate were prepared using the sameprocedures outline above with the respective isomer replacing thetBoc-l-alanate used in Example 21 a).

d) Camptothecin-20-O-Glycinate, TFA Salt--Compound 47:

Referring now to FIG. 14, it can be seen that thecamptothecin-20-O-glycinate was prepared using a procedure similar toExample 21 a) above with the t-Boc-glycinyl camptothecin (46) replacingthe t-Boc-l-alanyl camptothecin (21) to give 47.

e) 10,11-Methylenedioxycamptothecin 20-O-glycinate TFA Salt

The process of Example 21, steps a)-d) are repeated using10,11-methylenedioxycamptothecin to form the 20-O-alanate, and theglycinate TFA salt.

Example 22

Camptothecin-20-O-ester of PEG_(40kDa) L-Alanine--Compound 25:

Method A:

I) PEG (40 kDa) dicarboxylic acid

a) Di-t-BUTYL ESTER OF PEG (40,000) DI-CARBOXYLIC ACID

A solution of 50 grams (1.3 mmoles) of PEG-(OH)₂ in 750 ml of toluenewas azeotroped with the removal of 150 ml of distillate. The reactionmixture was then cooled to 30° C., followed by the addition of 4 ml (4.0mmoles) of a 1.0 molar solution of potassium t-butoxide in t-butanol.The resulting mixture was stirred for 1 hour at room temperature,followed by the addition of 1.6 grams (8.0 mmoles) oft-butylbromoacetate. The resulting cloudy mixture was heated to reflux,followed by removal of the heat, and stirring for 18 hours at roomtemperature. The reaction mixture was filtered through celite and thesolvent removed by rotary evaporator. The residue was recrystallizedfrom methylene chloridelethyl ether to yield 45.2 grams (86% yield). Thenamed product, however was found to be over 99% pure, the startingmaterial being present in an amount of less than 1.0%. ³ CNMRassignments: (CH₃)₃ C, 27.7 ppm; (CH)₃ C, 80.9 ppm; C═O, 169.1 ppm.

b) PEG (40,000) DI-CARBOXYLIC ACID

A solution of 20.0 grams (0.5 mmoles) of PEG (40,000) carboxylic acidt-butyl ester, 100 ml of trifluoroacetic acid, and 0.1 ml of water in200 ml of methylene chloride was stirred at room temperature for 3hours. The solvent was then removed by rotary evaporation, followed byrecrystallization of the residue from methylene chloride/ethyl ether toyield 16.9 grams (84% yield) of product. Purity of the named product wasconfirmed to be in excess of 99%. ¹³ CNMR assignments: C═O, 170.9 ppm.

IIa) Synthesis of the Camptothecin-20-O-Alanate PEG Derivative

Referring to FIG. 6, PEG_(40kDa) diacid (6.5 g, 0.62 mmol) was dissolvedin 60 mL of anhydrous methylene chloride at room temperature and to thissolution at 0° C. were added DIPC (148 μL, 0.97 mmol), DMAP (296 mg,2.43 mmol) and compound 23 (627 mg, 0.97 mmol). The reaction mixture wasallowed to warm to room temperature and left for 16 hours. The solutionwas washed with 0.1N HCl, dried and evaporated under reduced pressure toyield 25 as a white solid which was recrystallized from 2-propanol (5.5g, 83.%). ¹³ C and ¹ H NMR analysis confirmed the structure. ¹³ C NMR(CDCl₃) δ 6.81, 16.93, 30.80, 46.59, 49.28, 66.17, 69.77, 70.2-71(PEG),76.53, 94.79, 119.20, 127.18, 127.53, 127.91, 128.95, 129.72, 130.68,144.58, 145.76, 148.05, 151.46, 156.37, 165.99, 168.87, 170.32.

The procedure of IIa) was repeated using propane phosphonic acid cyclicanhydride (PPACA) in place of DIPC in order to form the named compound.

The racemic mixture is prepared in the same manner.

IIb) Synthesis of the Camptothecin-20-O-Glycinate PEG_(40kDa) -AmideDerivative--Compound 48:

Referring now to FIG. 14, the camptothecin-20-O-ester of PEG_(40kDa)glycinate was prepared using a procedure similar to that illustrated inExample 22a with compound 47 (FIG. 14) replacing compound 23 (FIG. 6) inorder to provide compound 48

III) Analysis of Camptothecin 20-O-ester of PEG_(40kDa) l-Alanine(25):

The UV absorbance of native camptothecin in methylene chloride wasdetermined at 227 nm for five different concentrations ranging from 4 to21 μM. From the standard plot of absorbance vs. concentration, theabsorption coefficient for camptothecin was calculated to be 2.96×10⁴Mol⁻¹ Cm⁻¹. Camptothecin compound 25 was dissolved in methylene chlorideat an approximate concentration of 4 μM, and the UV absorbance of thiscompound at 227 nm was determined. Using this value, and employing theabsorption coefficient obtained from above, the concentration ofcamptothecin in the sample was determined. Thus, dividing this value bythe camptothecin-PEG ester concentration provided the percentage ofcamptothecin in the esters.

Determination of % of camptothecin in the product using the UV methodindicated 2 eq. of camptothecin per PEG molecule.

IV) Synthesis of the Camptothecin-20-O-Glycinate PEG_(40kDa) CarbamateDerivative--Compound 49:

Referring now to FIG. 15, PEG_(40kDa) di-SC-PEG (2.0 g, 0.59 mmol)prepared according to the method described in U.S. Pat. No. 5,122,614,the contents of which are incorporated herein by reference, wasdissolved in 40 mL of anhydrous chloroform at room temperature. To thissolution was added DMAP (60.7 mg, 0.5 mmol) and compound 47 (122 mg, 0.2mmol). The reaction mixture was allowed to warm to room temperature andleft for 16 hours. The solution was washed with 0.1N HCl, dried andevaporated under reduced pressure to yield the title compound 49 as awhite solid which was recrystallized from 2-propanol.

Example 23

Camptothecin-20-O-ester of PEG_(40kDa) (d/l)-Alanine--Compound 25:

Method B:

Referring now to FIG. 9 for guidance, compound 25 can also be preparedusing a similar procedure to that shown below in Example 30 in order toprepare compound 31, substituting PEG-d-alanine 28, or PEG-l-alanine 29(shown in FIG. 7) in place of 27.

Example 24

Camptothecin-20-O-ester of PEG_(40kDa) (d)-Alanine--Compound 26:

Method A:

As shown in FIG. 6, the title compound is prepared in a similar manneras that used for preparing compound 25 in Example 22 usingtBoc-d-Alanine as starting material.

Example 25

Camptothecin-20-O-ester of PEG_(40kDa) (d)-Alanine--Compound 26:

Method B:

Compound 26 is also prepared using a similar procedure to that describedin Example 30 for preparing compound 31 and substituting PEG-d-Alanine29 (See FIG. 7) in place of 27. The racemic alanine mixture can also beprepared using either of the foregoing procedures.

Example 26

PEG_(40kDa) -β-Alanine(27):

As shown in FIG. 7, PEG_(40kDa) diacid (3 g, 0.075 mmol) was dissolvedin 30 mL of anhydrous methylene chloride at room temperature. To thissolution at 0° C. were added DIPC (91.4 μL 0.72 mmol), DMAP (128 mg,1.04 mmol) and β-alanine-t-butylester (109 mg, 0.59 mmol). The reactionmixture was allowed to warm to room temperature after 3 hours and leftfor 16 hours. The solution was washed with 0.1N HCl, dried andevaporated under reduced pressure to yield PEG_(40kDa) β-alanine-t-butylester as a white solid which was dissolved in a mixture of methylenechloride (50 ml) and trifluoroacetic acid (25 ml) at 0° C. forovernight. Solvent was removed and the solid was recrystallized frommethylene chloride/ether to give 27 (2.3 g, 77%).

¹³ C NMR (CDCl₃) δ: 32.99, 33.62, 68.10, 69.72, 169.08, 172.04.

Example 27

PEG_(40kDa) -d-Alanine(28):

As shown in FIG. 7, the title compound is prepared by using a similarprocedure to that used for synthesizing compound 27 in Example 26,substituting (d)-alanine-t-butyl ester in place of β-alanine-t-butylester.

Example 28

PEG_(40kDa) -l-Alanine(29):

The title compound is prepared by using a similar procedure to that usedfor synthesizing compound 27 in Example 26, substituting(l)-alanine-t-butyl ester in place of β-alanine-t-butyl ester. (See FIG.7).

Example 29

a) Paclitaxel-2'-O-ester of 27--Compound 30a:

Referring to FIG. 8, PEG_(40kDa) β-alanine (27, 2.3 g, 0.057 mmol) wasdissolved in 20 mL of anhydrous methylene chloride at room temperature.To this solution at 0° C. were added DIPC (32 μL, 0.2 mmol), DMAP (25mg, 0.2 mmol) and paclitaxel (175.6 mg, 0.2 mmol). The reaction mixturewas allowed to warm to room temperature and left for 16 hours. Thesolution was washed with 0.1N HCl, dried and evaporated under reducedpressure to yield 30a as a white solid (2 g, 87%) which wasrecrystallized from 2-propanol.

¹³ C NMR (CDCl₃) δ: 9.08, 14.22, 21.49, 21.89, 22.18, 25.9, 33.55,34.90, 35.03, 35.21, 42.67, 46.9, 52.22, 57.51, 67.59-71.96 (PEG),73.97, 74.60, 75.01, 80.11, 83.52, 126.32, 127.11, 127.57, -128.05,128.17, 128.65, 129.50, 130.79, 131.96, 133.06, 136.75, 141.84, 165.97,166.77, 167.45, 169.21, 169.70, 170.28, 170.33, 202.82.

b) Paclitaxel-2'-O-ester of 28--Compound 30b:

The procedure of Example 29a was repeated using d-alanine instead ofβ-alanine to yield compound 30b.

c) Paclitaxel-2'-O-ester of 29--Compound 30c:

The procedure of Example 29a was repeated using l-alanine instead ofβ-alanine to yield compound 30c.

Example 30

Camptothecin 20-O-ester of 27--Compound 31:

Referring now to FIG. 9, PEG_(40kDa) β-alanine(27, 2.3 g, 0.057 mmol) isdissolved in 20 mL of anhydrous methylene chloride at room temperatureand to this solution at 0° C. are added DIPC (32 μL, 0.2 mmol), DMAP (25mg, 0.2 mmol) and camptothecin (130 mg, 0.25 mmol). The reaction mixtureis allowed to warm to room temperature and left for 16 hours. Thesolution is washed with 0.1N HCl, dried and evaporated under reducedpressure to yield 31.

Example 31

Podophyllotoxin-4-O-ester of 27--Compound 32:

Referring now to FIG. 10, PEG_(40kDa) β-alanine (27, 2.3 g, 0.057 mmol)is dissolved in 20 mL of anhydrous methylene chloride at roomtemperature. To this solution at 0° C. are added DIPC (27.3 μL, 0.18mmol), DMAP (21.9 mg, 0.18 mmol) and podophyllotoxin (110 mg, 0.25mmol). The reaction mixture is allowed to warm to room temperature andleft for 16 hours. The solution is washed with 0.1N HCl, dried andevaporated under reduced pressure to yield 32 as a white solid which isrecrystallized from 2-propanol.

Example 32

Camptothecin-20-O-ester of Bromoacetic Acid--Compound 35:

Referring to FIG. 11, to a suspension of camptothecin, (1 g, 2.87 mmol)in CH₂ Cl₂ (700 ml) was added bromoacetic acid (33, 1.2 g, 8.61 mmol,Aldrich) diisopropylcarbodiimide (1.3 ml, 8.61 mmol) anddimethylaminopyridine (DMAP, 700 mg, 5.74 mmo) at 0° C. Stirring wascontinued for 4 hours. The resulting yellow solution was concentrated toabout 100 ml and washed with 1N hydrochloric acid (10 ml×2), followed by1% aqueous sodium bicarbonate solution (10 ml×2). The organic layer wasdried (anhyd.MgSO₄) and evaporated in vacuo to give a yellow solid whichwas recrystallized from ethyl acetate. The product was then trituratedwith methanol (10 ml), and the slurry filtered to yield 35 (0.9 g, 67%).

¹ H NMR(CDCl₃) δ 1.0(t), 1.84(s), 2.1-2.3(m), 3.94-4.4(q), 5.28(s),5.4-5.8(dd), 7.2(s), 7.27(s), 7.6-7.7(m), 7.81-7.87(m) 7.92-7.95(d),8.19-8.22(d), 8.39(s).

¹³ C NMR (CDCl₃) δ 7.52, 24.97, 31.77, 49.97, 67.16, 76.53, 95.73,120.29, 128.05, 128.17, 128.39, 129.64, 130.65, 131.17, 144.94, 146.48,148.84, 152.18, 157.24, 165.97, 166.83.

Example 33

Camptothecin-20-O-ester of Iodoacetic Acid--Compound 36:

Continuing to refer to FIG. 11, iodoacetic acid (34, 1.7 g, 9.13 mmol,Aldrich) diisopropylcarbodiimide (1.4 ml, 9.13 mmol) anddimethylaminopyridine (DMAP, 743 mg, 8.04 mmo) was added to a suspensionof camptothecin, (1.06 g, 3.04 mmol) in CH₂ Cl₂ (500 ml) at 0° C.Stirring was continued for 2 hours followed by 16 hours at roomtemperature. The resulting dark brown solution was concentrated to about100 ml and washed with 1N hydrochloric acid (10 ml×2), followed by 1%aqueous sodium bicarbonate solution (10 ml×2). The organic layer wasdried (anhyd.MgSO₄) and evaporated in vacuo to give a yellow solid whichwas recrystallized from ethyl acetate. The product was then trituratedwith methanol (10 ml), and the slurry filtered to yield 36 (1.3 g, 80%).

¹ H NMR(CDCl₃) δ 1.0(t), 2.1-2.3(m), 3.3 (s), 3.9-4.4(q), 5.28(s),5.4-5.8(dd), 7.2(s), 7.27(s), 7.6-7.7(m), 7.81-7.87(m)7.92-7.95(d),8.19-8.22(d), 8.39(s). ¹³ C NMR (CDCl₃) δ 7.52, 31.77, 49.97, 67.16,76.53, 95.73, 120.29, 128.05, 128.17, 128.39, 129.64, 130.65, 131.17,144.94, 146.48, 148.84, 152.18, 157.24, 165.97, 166.83.

Example 34

Reaction of 36 with PEG Diamine Hydrochloride(13)-Bis-Camptothecin-N-PEG_(40kDa) -20-O-Glycinate--Compound 37a:

Continuing to refer to FIG. 11, a solution of PEG_(40kDa) diaminehydrochloride (13, 5 g, 0.125 mmol), 20-Iodoacetyl camptothecin (36, 322mg, 0.624 mmol) and triethylamine (208 μL, 1.497 m.mol) in anhydrousmethylene chloride (75 mL) were stirred at room temperature for 3 days.The solvent was evaporated under reduced pressure and the solid obtainedwas recrystallized from DMF followed by 2-propanol to give 37a as awhite solid(4.4 g, 87.4%).

Example 35

Reaction of Compound 37a with AceticAnhydride-Bis-Camptothecin-N-Acetyl-PEG_(40kDa) -20-O-Glycinate-Compound37b:

Continuing to refer to FIG. 11, the preparation of the N-acetylderivative of 37a is illustrated and designated 37b. A solution ofcompound 37a (300 mg, 0.007 m.mol), acetic anhydride (28 μL) andpyridine (28 μL) in anhydrous methylene chloride (5 mL) was stirred atroom temperature for 18 hours. The solvent was removed and the residuewas crystallized from 2-propanol to give 200 mg of 37b.

Example 36

Reaction of 36 with PEG N-methyldiamine Hydrochloride (12)--Compound 38:

Also shown in FIG. 11, a solution of PEG_(40kDa) N-methyidiaminehydrochloride (12, 5 g, 0.125 mmol), 20-Iodoacetyl camptothecin (36, 322mg, 0.624 mmol) and triethylamine (208pL, 1.497m.mol) in anhydrousmethylene chloride (75 mL) were stirred at room temperature for 3 days.The solvent was evaporated under reduced pressure and the solid obtainedwas recrystallized from DMF followed by 2-propanol to give 38 as a whitesolid (4.5 g, 90%).

Example 37

20-Sarcosine Camptothecin (41):

Referring now to FIG. 12, a mixture of Sarcosine (5 g, 56.13 mmol), Bocanhydride (14.7 g, 67.35 m.mol) and sodium hydroxide (4.5 g, 112.26m.mol) in water (25 mL) was stirred at room temperature for 18 hours.The reaction mixture was cooled to 0° C. and was acidified to pH 3 with6N HCl and extracted with ethyl acetate. Evaporation of the solvent gaveBoc Sarcosine, 39, as a clear oil.

¹ H NMR (CDCl₃) δ 4.58(m), 3.0(s)4.0(m).

Boc-Sarcosine (39, 1.63 g, 8.61 mmol) was dissolved in 100 mL ofanhydrous methylene chloride at room temperature and to this solution at0° C. were added DIPC (1.3 mL, 8.61 mmol), DMAP (725 mg, 5.74 mmol) andcamptothecin (1 g, 2.87 mmol) at 0° C. The reaction mixture was allowedto warm to room temperature and left for 2 hours. The solution waswashed with 0.1N HCl, dried and evaporated under reduced pressure toyield a white solid which was recrystallized from 2-propanol to give20-Boc-sarcosine camptothecin (40, 750 mg, 50.3%).

20-Boc-sarcosine camptothecin (40, 750 mg) was dissolved in methylenechloride (4 ml) and trifluoroaceticacid (4 ml) and stirred at roomtemperature for 1 hour. Ether (10 ml) was added and the precipitatedsolid was filtered and dried to give 41 (550 mg, 85%) as yellow solid.

¹ H NMR(DMSO) δ 1.0(t), 2.2(m), 2.7 (s), 2.84(s), 4.4-4.6(dd), 5.11(brs), 5.34(s), 5.65(s), 7.36(s), 7.6-8.3(m), 8.74(s), 9.46(s).

¹³ C NMR (DMSO) δ 7.55, 30.21, 32.49, 47.87, 50.22, 66.40, 77.72, 95.34,118.84, 127.74, 127.95, 128.76, 129.67, 130.51, 131.65, 144.64, 147.88,152.24, 156.48, 158.23, 166.78.

Example 38

Reaction of 41 with PEG Dicarboxylic Acid--Compound 42:

Continuing to refer to FIG. 12, PEG_(40kDa) diacid (2 g, 0.05 mmol) wasdissolved in 30 mL of anhydrous methylene chloride at room temperatureand to this solution at 0° C. were added DIPC (30 μL, 0.20 mmol), DMAP(24 mg, 0.20 mmol) and 20-sarcosine camptothecin (41, 112 mg, 0.21mmol). The reaction mixture was allowed to warm to room temperature andleft for 16 hours. The solution was evaporated under reduced pressure toyield a white solid which was recrystallized from 2-propanol to give 42(1.4 g, 69%).

Example 39

a) PEG_(40kDa) Glycine (50):

PEG_(40kDa) diacid (9.5 g, 0.23 mmol) was dissolved in 20 mL of anhyd.methylene chloride at room temperature and to this solution at 0° C.were added DIPC (141 μL, 0.92 mmol), DMAP (197 mg, 1.6 mmol) andglycine-t-butylester (176.4 mg, 0.92 mmol) at 0° C. The reaction mixturewas allowed to warm to room temperature after 3 hours and left for 16hours. The solution was washed with 0.1N HCl, dried and evaporated underreduced pressure to yield PEG_(kDa) glycine t-butyl ester as a whitesolid which was dissolved in a mixture of methylene chloride (50 ml) andtrifluoroaceticacid (25 ml )at 0° C. for overnight. Solvent was removedand the solid was recrystallized from methylene chloride/ether to give50 (7.1 g, 75%). ¹³ C NMR (CDCl₃) δ 39.42, 69.59, 70.19, 169.39, 169.46.

b) PEG_(40kDa) Phenylalanine (51):

PEG_(40kDa) diacid (9.5 g, 0.23 mmol) was dissolved in 20 mL of anhyd.methylene chloride at room temperature and to this solution at 0° C.were added DIPC (141 μL, 0.92 mmol), DMAP (197 mg, 1.6 mmol) andphenylalanine-t-butyl ester (176.4 mg, 0.92 mmol) at 0° C. The reactionmixture was allowed to warm to room temperature after 3 hours and leftfor 16 hours. The solution was washed with 0.1N HCl, dried andevaporated under reduced pressure to yield PEG_(40kDa) phenylalaninet-butyl ester as a white solid which was dissolved in a mixture ofmethylene chloride (50 ml) and trifluoroaceticacid (25 ml ) at 0° C. forovernight. Solvent was removed and the solid was recrystallized frommethylene chloride/ether to give 51 (7.1 g, 75%).

¹³ C NMR (CDCl₃) δ 39.42, 69.59, 70.19, 169.39, 169.46.

c) PEG_(40kDa) Leucine (52):

PEG_(40kDa) diacid (9.5 g, 0.23 mmol) was dissolved in 20 mL of anhyd.methylene chloride at room temperature and to this solution at 0° C.were added DIPC (141 μL, 0.92 mmol), DMAP (197 mg, 1.6 mmol) andleucine-tbutyl ester(176.4 mg, 0.92 mmol) at 0° C. The reaction mixturewas allowed to warm to room temperature after 3 hours and left for 16hours. The solution was washed with 0.1N HCl, dried and evaporated underreduced pressure to yield PEG_(40kDa) leucine t-butyl ester as a whitesolid which was dissolved in a mixture of methylene chloride (50 ml)andtrifluoroaceticacid (25 ml) at 0° C. for overnight. Solvent was removedand the solid was recrystallized from methylene chloride/ether to give52 (7.1 g, 75%). ¹³ C NMR (CDCl₃) δ 39.42, 69.59, 70.19, 169.39, 169.46.

d) PEG_(40kDa) Proline (53):

PEG_(40kDa) diacid (9.5 g, 0.23 mmol) was dissolved in 20 mL of anhyd.methylene chloride at room temperature and to this solution at 0° C.were added DIPC (141 μL, 0.92 mmol), DMAP (197 mg, 1.6 mmol) andproline-t-butylester (176.4 mg, 0.92 mmol) at 0° C. The reaction mixturewas allowed to warm to room temperature after 3 hours and left for 16hours. The solution was washed with 0.1N HCl, dried and evaporated underreduced pressure to yield PEG_(40kDa) proline t-butyl ester as a whitesolid which was dissolved in a mixture of methylene chloride(50 ml)andtrifluoroaceticacid (25 ml )at 0° C. for overnight. Solvent was removedand the solid was recrystallized from methylene chloride/ether to give53 (7.1 g, 75%). ¹³ C NMR (CDCl₃) δ 39.42, 69.59, 70.19, 169.39, 169.46.

e) PEG_(40kDa) Methionine (54):

PEG_(40kDa) diacid (9.5 g, 0.23 mmol) was dissolved in 20 mL of anhyd.methylene chloride at room temperature and to this solution at 0° C.were added DIPC (141 μL, 0.92 mmol), DMAP (197 mg, 1.6 mmol) andmeththionine-t-butylester (176.4 mg, 0.92 mmol) at 0° C. The reactionmixture was allowed to warm to room temperature after 3 hours and leftfor 16 hours. The solution was washed with 0.1N HCl, dried andevaporated under reduced pressure to yield PEG_(40kDa) methioninet-butyl ester as a white solid which was dissolved in a mixture ofmethylene chloride (50 ml) and trifluoroaceticacid (25 ml )at 0° C. forovernight. Solvent was removed and the solid was recrystallized frommethylene chloridelether to give 54 (7.1 g, 75%). ¹³ C NMR (CDCl₃) δ39.42, 69.59, 70.19, 169.39, 169.46.

Example 40

Acyclovir-PEG Prodrug:

PEG_(40kDa) L-alanine diacid (29, 11.5 g, 0.287 mmol) is dissolved in200 mL of anhydrous methylene chloride at room temperature and to thissolution at 0° C. are added DIPC (0.175 ml, 1.15 mmol μL), DMAP (140 mg,1.15 mmol) and acyclovir (258 mg, 1.15 mmol). The reaction mixture isallowed to warm to room temperature after 2 hours and left for 16 hours.The solution is concentrated to about 100 ml and filtered through celiteand the filterate is evaporated under reduced pressure to yieldacyclovir-PEG prodrug as a solid which is recrystallized from CH₂ Cl₂/ether.

Example 41

CyclosporinA-PEG Prodrug:

PEG_(40kDa) glycine diacid (50, 11.5 g, 0.287 mmolg,) is dissolved in200 mL of anhydrous methylene chloride at room temperature and to thissolution at 0° C. are added DIPC (0. 175 ml, 1.15 mmol μL), DMAP (140mg, 1.15 mmol) and cyclosporin A (1.38 g, 1.15 mmol). The reactionmixture is allowed to warm to room temperature after 2 hours and leftfor 16 hours. The solution is concentrated to about 100 ml and filteredthrough celite and the filterate is evaporated under reduced pressure toyield cyclosporin A-PEG prodrug as a solid which is recrystallized fromCH₂ Cl₂ /ether.

Example 42

Amoxicillin-PEG Prodrug:

PEG_(40kDa) phenylalanine diacid (51, 11.5 g, 0.287 mmol) is dissolvedin 200 mL of anhydrous methylene chloride at room temperature and tothis solution at 0° C. are added DIPC (0.175 ml, 1.15 mmol μL), DMAP(140 mg, 1.15 mmol) and amoxicillin (419 mg, 1.15 mmol). The reactionmixture is allowed to warm to room temperature after 2 hours and leftfor 16 hours. The solution is concentrated to about 100 ml and filteredthrough celite and the filterate is evaporated under reduced pressure toyield amoxicillin-PEG prodrug as a solid which is recrystallized fromCH₂ Cl₂ /ether.

Example 43

Fluconazole-PEG Prodrug:

PEG_(40kDa) leucine diacid (52, 11.5 g, 0.287 mmol) is dissolved in 200mL of anhydrous methylene chloride at room temperature and to thissolution are added DIPC (0.175 ml, 1.15 mmol μL), DMAP (140 mg, 1.15mmol) and fluconazole (352 mg, 1.15 mmol) at 0° C. The reaction mixtureis allowed to warm to room temperature after 2 hours and left for 16hours. The solution is concentrated to about 100 ml and filtered throughcelite and the filterate is evaporated under reduced pressure to yieldfluconazole-PEG prodrug as a solid which is recrystallized from CH₂ Cl₂/ether.

Example 44

Floxuridine-PEG Prodrug:

PEG_(40kDa) proline diacid (53, 0.5 g, 0.0125 mmol,) is dissolved in 20mL of anhydrous methylene chloride at room temperature and to thissolution are added 2-chloro-1-methylpyridinium iodide (17 mg, 0.067mmol), DMAP (17 mg, 0.14 mmol) and floxuridine (13 mg, 0.049 mmol) at 0°C. The reaction mixture is allowed to warm to room temperature after 2hours and left for 16 hours. The solution is concentrated to about 100ml and filtered through celite and the filterate is evaporated underreduced pressure to yield floxuridine-PEG prodrug as a solid which isrecrystallized from CH₂ Cl₂ /ether.

Example 45 IN VITRO BIOASSAY

In this example, a series of in vitro assays were conducted to determinethe IC₅₀ for unmodified camptothecin, unmodified paclitaxel and severalof the high molecular weight prodrugs prepared as set forth above.

All compounds were independently tested against one or more of theP388/0 (murine lymphoid neoplasm, Southern Research Institute), HT-29(human colon carcinoma) and A549 (human lung adeno carcinoma) celllines.

The P388/0 cells were grown in RPMI 1640 medium (Whittaker Bioproducts,Walkersville, Md.)+10% FBS (Hyclone Inc., Logan Utah). The HT-29 cellswere grown in DMEM (GIBCOBRL)+10% FBS (Hyclone, Inc.). The A549 cellswere grown in DMEM/F-12 (Biowhitaker)+10% FBS (Heat inactivated).Bioassays were performed in their respective media containingantibiotics and fungizone.

Camptothecin and paclitaxel were dissolved in DMSO and diluted to theappropriate concentration in culture media. The PEG-Camptothecin andPEG-paclitaxel prodrugs were dissolved in water and diluted to theappropriate concentrations in culture media.

The assays were performed in duplicate in 96-well microtiter cellculture plates. Two fold serial dilution of the compounds were done inthe microtiter plates. Cells were detached by incubating with 0.1%Trypsin/Versene at 37°. Trypsin was inactivated by adding theappropriate media for each cell line containing 10% FBS. To each well ofthe microtiter plates, 10,000 cells were added. After three days, cellgrowth was measured by addition of a metabolic indicator dye, AlamarBlue, according to the manufacturer's protocol. The IC₅₀ value for eachtest compound was determined and compared to the IC₅₀ for theappropriate reference compound.

    ______________________________________                                        IC.sub.50 (nM)                                                                Compound #       P388   HT-29                                                 ______________________________________                                        Camptothecin      5     21                                                    Topotecan        29     99                                                    5a                7     30                                                    5b               18     153                                                   31               98     305                                                   37a              24     110                                                   37b              49     137                                                   42               15     46                                                    45                9     27                                                    49               33     40                                                    ______________________________________                                         P-388  Murine leukemia cell line                                              HT29  Human colon carcinoma cell line                                    

Referring now to the table, it can be seen that the relatively highmolecular weight polymer prodrugs compare favorably to unmodified formsof the drug.

Example 46 IN VIVO STUDY

In this Example, the in vivo activity of some of the compounds preparedin accordance with the present invention was assessed using the MurineLeukemia Model and the Colorectal Xenograft model.

Murine Leukemia Model (In vivo P388)

The compounds shown in the Table below were screened for in vivoactivity against the murine leukemia cell line P388/0 (mouse, lymphoidneoplasm). The cell line was obtained from Southern Research Institute(Birmingham, Ala.) and grown in RPMI 1640 supplemented with 10% FBS.P388/0 cells were subcultured two times per week and log phase cultures(viability ≧95%/o) were used for all in vivo experiments. Female CD2F1mice (Taconic Farms, Germantown, N.Y.) at 7-8 weeks of age were used forstudy. Following one week of acclimation, mice were implanted ip withP388/0 cells (5×10⁵ cells/mouse) at designated day 0 (zero). The micewere randomly assigned to experimental groups (10-20 per group). Thegroups included Control groups and several which received one of thedrugs or prodrugs. The mice were then dosed (500 μL, ip) for 5consecutive days (days 1-5). Control groups received vehicle (intralipidor water). The mice were monitored for up to 40 days, and the treatmentwas evaluated and expressed as the percentage survival at 40 days.

Colorectal Xenograft (In vivo HT-29)

Female nu/nu mice (Harlan Sprague Dawley, Madison, Wis.), 18-24 g and10-14 weeks old, at onset of treatment were used. The solid tumor HT-29(human, colon adenocarcinoma) was obtained from the ATCC (HTB 38) andgrown in DMEM supplemented with 10% FBS. Cells were subcultured once aweek and for in vivo experiments viabilities were ≧90%. Mice were housedin microisolator filtration racks, and maintained with filteredacidified water and sterile laboratory chow ad libitum. Following oneweek of acclimation, tumors were established by injecting 1×10⁶harvested HT-29 tumor cells in a single subcutaneous site, on the flankof mice in the left axillary region. The tumor injection site wasobserved twice weekly and measured once palpable. The tumor volume foreach mouse was determined by measuring two dimensions with calipers andcalculated using the formula: tumor volume=(length×width²)/2. Whentumors reached the average volume of 300 mm³, the mice were divided intotheir experimental groups. The non-Control groups received Camptothecin2.5 mg/kg/day, the prodrugs being dosed on the basis of camptothecincontent. The mice were sorted to evenly distribute tumor size, groupedinto 5 mice/cage, and ear punched for permanent identification. Micereceiving drugs were treated i.p. 5 times a week, Monday through Fridayfor 5 weeks with 500 μL of test article. Mouse weight and tumor sizewere measured at the beginning of study and weekly through week 7. Theoverall growth of tumors was expressed as the percent change in tumorsize calculated by subtracting mean initial tumor volume from mean tumorvolume at the end of the treatment and dividing by mean initial tumorvolume. Thus, any tumor group which did not respond to treatment andgrew over the course of the experiment would display a zero or positivepercent change and treatment groups in which tumors regressed wouldexhibit a negative percent change.

The resulting data are presented below:

P388 data

    ______________________________________                                                      total active                                                                             50% survival                                                                             Survival                                  Compound #    dose mg/kg (days)     Rate %                                    ______________________________________                                        Control- untreated                                                                           0         13         NA                                        Camptothecin  16         NA         80                                        Camptothecin-20-O-ester of                                                                    11.4     21         10                                        PEG.sub.20kDa diacid                                                          Compound 15 (PEG.sub.40kDa)                                                                 16         NA         60                                        Compound 48 (PEG.sub.40kDa)                                                                 16         NA         80                                        ______________________________________                                    

The data in this Table illustrates that cure rate obtained using theprodrugs of the present invention was comparable to that of theunmodified or native camptothecin compound. Furthermore, groups treatedwith each of the prodrug compositions exceeded the 50% survival time ofthe untreated group. In addition, the survival rate of the highermolecular weight compounds was approximately the same as that with theunmodified camptothecin.

HT-29 data WEEK 5 (end of treatment)

    ______________________________________                                                Total active                                                                            Tumor growth                                                                             Body weight                                                                           Mortality                                Compound #                                                                            dose (mg/kg)                                                                            (%)        change (%)                                                                            (%)                                      ______________________________________                                        Control NA        723        +6      100                                      Campto- 62.5      -20        -3      50                                       thecin                                                                        15      62.5      -60         1      30                                       48      62.5      -80        -9       0                                       ______________________________________                                    

WEEK 7 (2 week post treatment)

    ______________________________________                                                  Total active                                                                              Tumor growth                                                                             Body weight                                  Compound #                                                                              dose (mg/kg)                                                                              (%)        change (%)                                   ______________________________________                                        Control   NA          1347       +8                                           Camptothecin                                                                            62.5         62        -3                                           15        62.5        -73        +13                                          48        62.5        -96        +25                                          ______________________________________                                    

In addition to the increased water solubility provided by the prodrugformulations of the present invention, the data indicates that thePEG-prodrug compounds are more efficacious and less toxic than parentcompounds. Of particular interest are the facts that even 2 weeks aftertreatment was ceased, the animals treated with the prodrugs stillexhibited decreases in tumor volume and the animals had body weightgains comparable to the control animals. While Applicants are not boundby theory, it is believed that the unique combination of highermolecular weight polymer and the controlled rate of hydrolysis of theparticular ester linkages allow therapeutic amounts of the parentcompound to be generated before the prodrug is cleared from the body. Itis also concluded that the prodrug compositions accumulated to a certaindegree in the tumor areas and provided a localized and residual effect.

Example 47

Preparation of Campltothecin-20-O-carbonylimidazolylcarbonate (54)

In a 100 mL round bottomed flask were placed camptothecin 1 (1 g, 2.88mmol), N,N-carbonyldidmidazole (2.335 g, 14.4 mmol) and methylenechloride (50 mL), stirred for 10 minutes, followed by the addition ofdimethylaminopyridine (110 mg, 0.864 mmol). The resulting clear solutionwas stirred at room temperature under a nitrogen atmosphere for 4 hours.The reaction mixture was diluted with 50 mL of methylene chloride,washed with 50 mL of 0.1 N HCl, followed by 50 mL of water. The organiclayer was dried over anhydrous magnesium sulfate, and evaporated todryness. The crude product thus obtained (604 mg, 95%) was used withoutfurther purification for the preparation of camptothecin 20-O-carbamatederivatives.¹³ C NMR (67.8 MHz, CDCl₃) δ: 166.21, 157.07, 151.91,148.75, 147.12, 146.83, 144.40, 137.10, 131.20, 131.09, 130.71, 129.466,128.33, 128.10, 120.11, 117.17, 95.13, 78.93, 67.17, 49.98, 31.82, 7.62.¹ H NMR (270 MHz, CDCl₃) δ: 8.41 (s, 1), 8.22 (s, 1), 8.20 (d, 1), 7.95(d, 1), 7.83 (t, 1), 7.67 (t, 1), 7.46 (s, 1), 7.25 (s, 1), 7.13 (s, 1),5.62 (d of d, 2), 5.32 (s, 2), 2.38 (m, 2), 1.10 (t, 3).

Example 48

Preparation of Camptothecin-20-O-para Nitrophenyl Carbonate (55)

In a 100 mL round bottomed flask were placed camptothecin 1 (0.5 g, 1.44mmol), para nitrophenylchloroformate (0.871 g, 4.3 mmol) and methylenechloride (15 mL), stirred for 30 minutes at -8° C., followed by theaddition of dimethylaminopyridine (1.06 g, 8.61 mmol). The resultingclear solution was stirred at room temperature under a nitrogenatmosphere for 2 hours. The precipitated solid was filtered and dried togive the product The filtrate was diluted with 25 mL of methylenechloride, washed with 10 mL of 0.1 N HCl, followed by 10 mL of 0.1 Nsodium bicarbonate solution. The organic layer was concentrated to 5 mLand precipitated with ether and over anhydrous. magnesium sulfate, andevaporated to dryness to give additional quantity of the product. Totalyield of the product (604 mg, 95%). ¹³ C NMR (67.8 MHz, CDCl₃) δ:166.80, 157.18, 155.05, 152.17, 151.23, 148.93, 146.83, 145.60, 144.82,131.31, 130.86, 129.59, 128.46, 128.25, 125.25, 121.70, 120.40, 95.47,79.32, 67.21, 50.05, 31.92, 7.64.

¹ H NMR (270 MHz, CDCl₃) δ: 8.43 (s, 1), 8.25 (d, 1), 8.22 (d, 2), 7.96(d, 1), 7.86 (t, 1), 7.70 (t, 1), 7.40 (d, 2), 7.38 (s, 1), 5.57 (d ofd, 2), 5.31 (s, 2), 2.31 (m, 2), 1.07 (t, 3).

Example 49

Preparation of Camptothecin-20-O-N-hydroiysuccinimidyl Carbonate (56)

In a 50 mL round bottomed flask were placed camptothecin 1 (100 mg,0.288 mmol), disuccinimidyl carbonate (0.369 mg, 1.44 mmol) andmethylene chloride (7 mL), stirred for 5 minutes, followed by theaddition of dimethylaminopyridine (110 mg, 0.864 mmol). The resultingclear solution was stirred at room temperature under a nitrogenatmosphere for 2 days. The reaction mixture was diluted with 50 mL ofmethylene chloride, washed with 25 mL of 0.1 N HCl, followed by 25 mL ofwater. The organic layer was dried over anhydrous. magnesium sulfate,and evaporated to dryness to give the product (60 mg, 95%). ¹³ C NMR(67.8 MHz, CDCl₃) δ: 168.53, 167.77, 160.28, 157.18, 153.00, 151.00,149.04, 144.03, 131.05, 130.63, 130.01, 128.38, 128.23, 128.13, 120.50,95.68, 80.88, 67.04, 50.08, 31.85, 20.17, 7.64.

¹ H NMR (270 MHz, CDCl₃) δ: 8.40 (s, 1), 8.29 (d, 1), 7.92 (d, 1), 7.84(t, 1), 7.67 (t, 1), 7.45 (s, 1), 5.53 (d of d, 2), 5.30 (s, 2), 3.10(t, 4), 2.32 (m, 2), 1.05 (t, 3).

Example 50a

Preparation of Camptothecin-20-O-PEG Carbamates (57).

A solution of 54 (176 mg, 0.4 mmol), and PEG 40 kDa NH₂ (4 g, 0.1 mmol)in anhydrous 2-propanol (75 mL ) and dimethylaminopyridine (37 mg, 0.3mmol ) was refluxed for 21 hours. The reaction mixture was cooled toroom temperature and the separated solid was filtered and washed withether. The solid obtained was recrystallized from 2-propanol (80 mL )twice to give 3.67 g of product. ¹³ C NMR (67.8 MHz, CDCl₃) δ: 172.0,162.0, 155.0, 153.0, 149.0, 145.0, 144.0, 134.0, 133.5, 133.0, 132.0,129.5, 128.5, 127.5, 97.5, 88.5, 68.03, 58.5, 50.00, 39.95, 32.0, 7.0.

Example 50b

Preparation of Cammptothecin-20-O-PEG-5000 Carbamate.

The process of Example 50a described supra was repeated except thatPEG-5000 was used in place of PEG 40 kDa NH₂. The T_(1/2) in rat plasmafor this compound was determined to be 7.5 hours.

Example 51a

Preparation of Camptothecin-20-O-PEG 40kDa Carbonate (58).

In a 25 ml round bottomed flask under nitrogen atmosphere were placedcamptothecin (228 mg, 0.65 mmol), PEG 40 kDa OH (4 g, 0.1 mmol) and 5 mLof anhydrous methylene chloride. To this solution were added triphosgene(72 mg, 0.24 mmol) and dimethylaminopyridine (243 mg, 1.9 mmol) andstirring continued for 16 hours. The reaction mixture was filteredthrough celite and evaporated to dryness. The solid obtained wascrystallized from 2-propanol. ¹³ C NMR (67.8 MHz, CDCl₃)

Example 51b

Preparation of Camptothecin-20-O-PEG-5000 Carbonate

The process of Example 51a described supra was repeated except thatPEG-5000 was used in place of PEG 40 kDa NH₂.

Example 52

Preparation of Camptotbecin-20-O-methylcarbonate (59).

A solution of 55 (500 mg, 0.976 mmol), in anhydrous methanol (15 mL ),and methylene chloride (15 mL) and dimethylaminopyridine (60 mg, 0.488mmol ) was stirred at room temperature for 2 hours. The reactionmtixture was washed with 3 mL of 0.1 N HCl, followed by 3 mL of 0.1 Nsodium bicarbonate solution, dried (anhydrous magnesium sulfate) andevaporated to a small volume (5 mL) followed by the addition of ether toprecipitate the product. The solid obtained was filtered and dried togive 59 (0.395 g, 83%) ¹³ C NMR (67.8 MHz, CDCl₃) δ: 167.06, 157.44,154.34, 152.64, 149.25, 146.57, 145.94, 130.92, 130.58, 129.95, 128.65,128.38, 128.13, 128.02, 120.77, 95.97, 77.96, 67.12, 55.32, 50.00,31.19, 7.54.

¹ H NMR (270 MHz, CDCl₃) δ: 8.41 (s, 1), 8.24 (d, 1), 7.96 (d, 1), 7.85(t, 1), 7.68 (t, 1), 7.34 (s, 1), 5.56 (d of d, 2), 5.30 (s, 2), 3.79(s, 3), 2.23 (m, 2), 1.01 (t, 3).

Example 53

Preparation of Camptothecin-20-O-isobutyryl Carbonate (60).

To a solution of camptothecin (500 mg, 1.44 mmol) anddimethylaminopyridine (1.5 mg, 0.012 mmol) in anhydrous methylenechloride (25 mL ), was added isobutyryl chlorofornate and the contentswere stirred at room temperature for 2 hours. The reaction mixture waswashed with 3 mL of 0.1 N HCl, followed by 3 mL of 0.1 N sodiumbicarbonate solution, dried (anhydrous magnesium sulfate) and evaporatedto give the product. ¹³ C NMR (67.8 MHz, CDCl₃) δ: 167.19, 157.44,153.92, 152.62, 149.22, 146.49, 146.12, 130.92, 130.57, 129.93, 128.64,128.35, 128.13, 128.00, 120.67, 96.01, 75.05, 67.09, 49.98, 31.13,27.86, 18.75, 7.59.

¹ H NMR (270 MHz, CDCl₃) δ: 8.40 (s, 1), 8.23 (d, 1), 7.95 (d, 1), 7.84(t, 1), 7.67 (t, 1) 7.35 (s, 1), 5.55 (d of d, 2), 5.29 (s, 2), 3.90 (m,2), 2.24 (m, 2), 1.98 (m, 1), 1.01 (t, 3), 0.94 (d, 6).

Example 54

The IC₅₀ values for some of the compounds formed in examples 47-53 weredetermined using the same process as that described above with regard toexample 45 and compared to native camptothecin. The results are providedbelow.

    ______________________________________                                        Compound #                                                                            Description             IC.sub.50 (nM)                                ______________________________________                                                camptothecin             7                                            54      camptothecin-20-O-carbonylimidazolyl                                                                  69                                                    carbonate                                                             55      camptothecin-20-O-paranitrophenyl carbonate                                                           365                                           56      camptothecin-20-O-N-hydroxysuccinimidyl                                                               1030                                                  carbonate                                                             57      camptothecin-20-O-PEG5kDa carbamate                                                                   912                                           59      camptothecin-20-O-methyl carbonate                                                                    11                                            60      camptothecin-20-O-isobutyryl carbonate                                                                29                                            ______________________________________                                    

The various publications, patents, patent applications and publishedations mentioned in this application are hereby incorporated byreference herein.

We claim:
 1. A composition comprising the formula: ##STR20## wherein: Dis a residue of a biologically active moiety selected from the groupconsisting of acyclovir, cyclosporin A, amoxicillin, fluconazole, andfloxuridine;X is an electron withdrawing group; Y and Y' areindependently O or S; (n) is zero (0) or a positive integer; R₁ and R₂are independently selected from the group consisting of H, C₁₋₆ alkyls,aryls, substituted aryls, aralkyls, heteroalkyls, substitutedheteroalkyls and substituted C₁₋₆ alkyls; R₃ is a substantiallynon-antigenic polymer, C₁₋₁₂ straight or branched alkyl or substitutedalkyl, C₅₋₈ cycloalkyl or substituted cycloalkyl, carboxyalkyl,carboalkoxy alkyl, dialkylaminoalkyl, phenylalkyl, phenylaryl or##STR21## R₄ and R₅ are independently selected from the group consistingof H, C₁₋₆ alkyls, aryls, substituted aryls, aralkyls, heteroalkyls,substituted heteroalkyls, and substituted C₁₋₆ alkyls or jointly form acyclic C₅ -C₇ ring.
 2. The composition of claim 1, wherein R₃ is asubstantially non-antigenic polymer having a capping group Z.
 3. Thecomposition of claim 2, wherein Z is selected from the group consistingof OH, C₁₋₄ alkyl moieties, or ##STR22## wherein D' is selected from thegroup consisting of D, biologically active moieties other than D,dialkyl ureas, C₁₋₄ alkyls and carboxylic acids.
 4. The composition ofclaim 1, wherein R₁ and R₂ are independently H, methyl or ethyl.
 5. Thecomposition of claim 1, wherein said substituted C₁₋₆ alkyl is selectedfrom the group consisting of carboxyalkyls, aminoalkyls, dialkylaminos,hydroxyalkyls, and mercaptoalkyls.
 6. The composition of claim 1,wherein X is selected from the group consisting of O, N(R₁), S, SO andSO₂.
 7. The composition of claim 1, wherein X is selected from the groupconsisting of O and N(R₁).
 8. The composition of claim 1, wherein (n) iszero (0), 1, or
 2. 9. The composition of claim 1, wherein Y and Y' areO.
 10. The composition of claim 1, wherein R₃ comprises a polyalkyleneoxide.
 11. The composition of claim 10, wherein said polyalkylene oxidecomprises polyethylene glycol.
 12. The composition of claim 10, whereinsaid polyalkylene oxide has a molecular weight of from about 20,000 toabout 80,000.
 13. The composition of claim 10, wherein said polyalkyleneoxide has a molecular weight of from about 25,000 to about 45,000. 14.The composition of claim 13, wherein said polyalkylene oxide has amolecular weight of from about 30,000 to about 42,000.
 15. Thecomposition of claim 11, wherein R₃ is selected from the groupconsisting of:--C(Y)--(CH₂)_(n) --(CH₂ CH₂ O)_(x) --R",--C(Y)--Y--(CH₂)_(n) --(CH₂ CH₂ O)_(x) --R", --C(Y)--NR₁ --(CH₂)_(n)--(CH₂ CH₂ O)_(x) --R", and --CHR₁ --(CH₂)_(n) --(CH₂ CH₂ O)_(x) --R",whereinR₁ is independently selected from the group consisting of H, C₁₋₆alkyls, aryls, substituted aryls, aralkyls, heteroalkyls, substitutedheteroalkyls and substituted C₁₋₆ alkyls; (n) is zero (0) or is apositive integer; Y is O or S; R" is a capping group or R₁ ; and (x)represents the degree of polymerization.
 16. A method of treatingmammals with prodrugs, comprising:administering to a mammal in need ofsuch treatment an effective amount of a composition of claim
 1. 17. Acompound comprising the formula: ##STR23## wherein: D is a residue ofbiologically active moiety;X is an electron with drawing group; Y and Y'are independently O or S; (n) is zero or a positive integer; R₁ and R₂are independently selected from the group consisting of H, C₁₋₆ alkyls,aryls, substituted aryls, aralkyls, heteroalkyls, substitutedheteroalkyls and substituted C₁₋₆ alkyls; R₃ is a substantiallynon-antigenic polymer, C₁₋₁₂ straight or branched alkyl or substitutedalkyl, C₅₋₈ cycloalkyl or substituted cycloalkyl, carboxyalkyl,carboalkoxy alkyl, dialkylaminoalkyl, phenylalkyl, phenylaryl or##STR24## R₄ and R₅ are independently selected from the group consistingof H, C₁₋₆ alkyls, aryls, substituted aryls, aralkyls, heteroalkyls,substituted heteroalkyls, and substituted C₁₋₆ alkyls or jointly form acyclic C₅ -C₇ ring.
 18. The compound of claim 17, wherein X is NR₁. 19.The compound of claim 17, wherein X is NH.
 20. The compound of claim 17,wherein R₃ comprises a polyalkylene oxide.
 21. The compound of claim 20,wherein said polyalkylene oxide comprises polyethylene glycol.
 22. Thecompound of claim 20, wherein said polyalkylene oxide has a molecularweight of from about 20,000 to about 80,000.
 23. The compound of claim22, wherein said polyalkylene oxide has a molecular weight of from about25,000 to about 45,000.
 24. The compound of claim 23, wherein saidpolyalkylene oxide has a molecular weight of from about 30,000 to about42,000.
 25. The compound of claim 17, wherein Y and Y' are O.
 26. Thecompound of claim 17, wherein D is a residue of a biologically moietyselected from the group consisting of paclitaxel, taxotere, camptothecinand podophyllotoxin.
 27. The compound of claim 17, wherein D is aresidue of a biologically active moiety selected from the groupconsisting of paclitaxel, taxane and taxotere; and Y' is attached to the2' position of said paclitaxel, taxane or taxotere residues.
 28. Thecompound of claim 17, wherein D is a camptothecin derivative residue andY' is attached to the 20 S position of said camptothecin derivative. 29.The compound of claim 17, wherein D is a residue selected from the groupconsisting of biologically active proteins, enzymes, peptides,anti-tumor agents, cardiovascular agents, anti-neoplastics,anti-infectives, anti-fungals, anti-anxiety agents, gastrointestinalagents, central nervous system-activating agents, analgesics, fertilityagents, contraceptive agents, anti-inflammatory agents, steroidalagents, anti-urecernic agents, vasodilating agents, and vasoconstrictingagents.
 30. A method of treating mammals with prodrugs,comprising:administering to a mammal in need of such treatment aneffective amount of a compound of claim
 17. 31. A method of preparing apolymeric conjugate, comprising:a) reacting a biologically activenucleophile with a bifunctional spacer-containing moiety in the presenceof a coupling agent; b) forming the trihaloacetic acid derivative of thebiologically active nucleophile containing the bifunctional spacercontaining moiety; and c) reacting the trihaloacetic acid derivative ofthe biologically active nucleophile containing the bifunctional spacercontaining moiety with a compound of the formula ##STR25## to form aconjugate of the formula ##STR26## wherein D is a residue of abiologically active moiety.